Nucleic acid amplification

ABSTRACT

The present disclosure provides methods, compositions, kits and systems for nucleic acid amplification. In some embodiments, nucleic acid amplification methods include subjecting the nucleic acid to be amplified to partially denaturing conditions. In some embodiments, nucleic acid amplification methods include amplifying without fully denaturing the nucleic acid that is amplified. In some embodiments, the nucleic acid amplification method employs an enzyme that catalyzes homologous recombination and a polymerase. In some embodiments, methods for nucleic acid amplification can be conducted in a single reaction vessel and/or in a single continuous liquid phase of a reaction mixture, without need for compartmentalization of the reaction mixture or immobilization of reaction components. In some embodiments, methods for nucleic acid amplification comprise amplifying at least one polynucleotide onto a surface under isothermal amplification conditions, optionally in the presence of a polymer which can include a sieving agent and/or a diffusion-reducing agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/442,341 filed on Jun. 14, 2019, which is a continuation of U.S.application Ser. No. 15/091,717 filed on Apr. 6, 2016 and issued as U.S.Pat. No. 10,329,544 on Jun. 25, 2019, which is a continuation of U.S.application Ser. No. 14/023,361 filed Sep. 10, 2013 and issued as U.S.Pat. No. 9,334,531 on May 10, 2016, which claims the benefit of U.S.Provisional Application Nos. 61/699,810, filed Sep. 11, 2012;61/767,766, filed Feb. 21, 2013; 61/781,016, filed Mar. 14, 2013;61/792,247, filed Mar. 15, 2013; 61/822,226, filed May 10, 2013;61/822,239, filed May 10, 2013; 61/858,977, filed Jul. 26, 2013;61/859,000, filed Jul. 26, 2013; and 61/876,136 filed Sep. 10, 2013.U.S. application Ser. No. 14/023,361 is a continuation-in-part of eachof the following applications: (1) U.S. application Ser. No. 13/923,232,filed Jun. 20, 2013 and issued as U.S. Pat. No. 9,371,557 on Jun. 21,2016, which claims benefit of U.S. Provisional Application Nos.61/692,830, filed Aug. 24, 2012; 61/699,810, filed Sep. 11, 2012;61/767,766, filed Feb. 21, 2013; 61/781,016, filed Mar. 14, 2013;61/792,247, filed Mar. 15, 2013; 61/822,226, filed May 10, 2013;61/822,239, filed May 10, 2013; which is also a continuation of U.S.application Ser. No. 13/842,296, filed Mar. 15, 2013 and issued as U.S.Pat. No. 9,309,557 on Apr. 12, 2016, which claims benefit of U.S.Provisional Application Nos. 61/635,584, filed Apr. 19, 2012;61/692,830, filed Aug. 24, 2012; 61/699,810, filed Sep. 11, 2012;61/767,766, filed Feb. 21, 2013; 61/781,016, filed Mar. 14, 2013;61/792,247, filed Mar. 15, 2013; which said application Ser. No.13/842,296 (filed Mar. 15, 2013) is a continuation-in-part of U.S.application Ser. No. 13/328,844, filed Dec. 16, 2011 and is nowabandoned, which claims benefit of U.S. Provisional Application Nos.61/424,599, filed Dec. 17, 2010; 61/445,324, filed Feb. 22, 2011;61/451,919, filed Mar. 11, 2011; 61/526,478, filed Aug. 23, 2011; and61/552,660, filed Oct. 28, 2011; which said application Ser. No.13/842,296 (filed Mar. 15, 2013) is also a continuation-in-part of U.S.application Ser. No. 13/828,049, filed Mar. 14, 2013 and issued as U.S.Pat. No. 9,309,566 on Apr. 12, 2016, which claims benefit of U.S.Provisional Application No. 61/692,830; filed Aug. 24, 2012, and whichis a continuation-in-part of U.S. application Ser. No. 13/328,844, filedDec. 16, 2011, which claims benefit of U.S. Provisional Application Nos.61/424,599, filed Dec. 17, 2010; 61/445,324, filed Feb. 22, 2011;61/451,919, filed Mar. 11, 2011; 61/526,478, filed Aug. 23, 2011; and61/552,660, filed Oct. 28, 2011; which said application Ser. No.13/842,296 (filed Mar. 15, 2013) is also a continuation-in-part of PCTInternational Application No. PCT/US2011/65535, filed Dec. 16, 2011,which claims benefit of U.S. Provisional Application Nos. 61/424,599,filed Dec. 17, 2010; 61/445,324, filed Feb. 22, 2011; 61/451,919, filedMar. 11, 2011; 61/526,478, filed Aug. 23, 2011; and 61/552,660, filedOct. 28, 2011; and (2) International PCT Application No.PCT/US2013/37352, filed Apr. 19, 2013, which claims priority to U.S.Provisional Application No. 61/635,584, filed Apr. 19, 2012. Each ofthese applications cited above is hereby incorporated by reference inits entirety.

Throughout this application various publications, patents, and/or patentapplications are referenced. The disclosures of these publications,patents, and/or patent applications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND

Nucleic acid amplification is very useful in molecular biology and haswide applicability in practically every aspect of biology, therapeutics,diagnostics, forensics and research. Generally, amplicons are generatedfrom a starting template using one or more primers, where the ampliconsare homologous or complementary to the template from which they weregenerated. Multiplexed amplification can also streamline processes andreduce overheads. This application relates to methods and reagents fornucleic acid amplification and/or analysis.

SUMMARY

Methods, reagents and products of nucleic acid amplification and/oranalysis are provided herein. Amplification can make use of immobilizedand/or soluble primers. A single set of primers can be mixed withdifferent templates, or a single template can be contacted with multipledifferent primers, or multiple different templates can be contacted withmultiple different primers. Amplicons generated from methods providedherein are suitable substrates for further analysis, e.g., sequencedetermination.

In some embodiments, the present teachings provide compositions,systems, methods, apparatuses and kits for nucleic acid amplification.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic showing an embodiment of template walking.In an alternative embodiment, the immobilized primer comprises anadenosine-rich sequence designated as (A)_(n), e.g., (A)₃₀, and theprimer binding site for the immobilized primer on the template comprisesa complementary T-rich sequence, e.g., (T)₃₀.

FIG. 2 depicts an overview of amplification on beads by template walkingand deposition of beads onto a planar array for sequencing.

FIG. 3 depicts some alternative embodiments using semiconductor-baseddetection of sequencing by synthesis. Template walking can be used togenerate a population of clonal amplicons on a bead or on the base orbottom of a reaction chamber. In an alternative embodiment, theimmobilized primer comprises an adenosine-rich sequence designated as(A)_(n), e.g., (A)₃₀, and the primer binding site for the immobilizedprimer on the template comprises a complementary T-rich sequence, e.g.,(T)₃₀.

FIG. 4 depicts some alternative embodiments of immobilization sites inthe form of primer lawns on planar substrates. Arrays of separatedimmobilization sites can be used or else a single continuous lawn ofprimers can be considered to be a random array of immobilization sites.Optionally, the location of one or more immobilization sites in thecontinuous lawn of primers can be undetermined as yet, where thelocation is determined at the time of attachment of the initial templatebefore walking or is determined by the space occupied by the amplifiedcluster. In an alternative embodiment, the immobilized primer comprisesan adenosine-rich sequence designated as (A)_(n), e.g., (A)₃₀, and theprimer binding site for the immobilized primer on the template comprisesa complementary T-rich sequence, e.g., (T)₃₀.

FIG. 5 illustrates the influence of temperature on the template walkingreaction. A graphical plot of the delta Ct before and after the templatewalking amplification was calculated and plotted against reactiontemperature.

FIG. 6 provides a table of the Ct values of the 96 duplex TaqMan qPCRreactions.

FIG. 7 depicts data illustrating roughly 100,000-fold amplification bytemplate walking on beads. Delta Ct before and after the templatewalking reaction and fold of amplification before and after the templatewalking reaction was calculated and plotted against reaction time.

FIGS. 8A-8C provide a schematic depiction of an exemplarystrand-flipping and walking strategy. FIG. 8A Template walking, FIG. 8BStrand flipping to generate flipped strands, FIG. 8C addition of newprimer-binding sequence Pg′ on final flipped strands.

FIG. 9 depicts an exemplary read length histogram from an Ion Torrent™PGM sequencing run of polynucleotide templates amplified using arecombinase-mediated amplification reaction.

FIG. 10 depicts an exemplary read length histogram from an Ion Torrent™Proton sequencing run of polynucleotide templates amplified using arecombinase-mediated amplification reaction.

FIG. 11 depicts an exemplary read length histogram from an Ion Torrent™Proton sequencing run of polynucleotide templates amplified using arecombinase-mediated amplification reaction.

FIG. 12 depicts an exemplary read length histogram from an Ion Torrent™Proton sequencing run of polynucleotide templates amplified using arecombinase-mediated amplification reaction.

FIG. 13 includes an illustration of an exemplary measurement system.

FIG. 14 includes an illustration of an exemplary measurement component.

FIG. 15 includes an illustration of exemplary array of measurementcomponents.

FIG. 16 includes an illustration of exemplary well configurations.

FIG. 17 includes an illustration of exemplary well and sensorconfigurations.

FIG. 18 , FIG. 19 , FIG. 20 and FIG. 21 include illustrations ofworkpieces during processing by an exemplary method.

FIG. 22 , FIG. 23 and FIG. 24 include illustrations of workpieces duringprocessing by an exemplary method.

FIG. 25 , FIG. 26 and FIG. 27 include illustrations of workpieces duringprocessing by an exemplary method.

FIG. 28 illustrates an exemplary block diagram of components of a systemfor nucleic acid sequencing according to an exemplary embodiment.

FIG. 29 illustrates an exemplary cross-sectional view of a portion ofthe integrated circuit device and flow cell according to an exemplaryembodiment.

FIG. 30 illustrates an exemplary cross-sectional view of representativechemical sensors and corresponding reaction regions according to anexemplary embodiment.

DETAILED DESCRIPTION

Conventional amplification of a nucleic acid template typically involvesrepeated replication of the template (and/or its progeny) usingappropriate synthetic systems. In such conventional methods, eachinstance of replication is typically preceded by denaturation of thetemplate to be amplified using extreme denaturing conditions, therebyrendering the template substantially single stranded. Some common andwidely used examples of extreme denaturing conditions used forconventional amplification including thermal denaturation usingtemperatures well above the melting point of the nucleic acid templateto be amplified (e.g., conventional PCR involving thermocycling usingdenaturation temperatures of well above 90° C., typically around 94-95°C.) or exposure of the template to harsh denaturants such as NaOH,guanidium agents, and the like. Such methods typically requirespecialized equipment (e.g., thermocyclers), and entail additionalmanipulations during the amplification process (e.g., annealing step forconventional PCR; wash step to remove chemical denaturants, etc.),thereby increasing the cost, effort and time associated with suchamplification, as well as constraining the yields ultimately obtainableusing such methods. Moreover, such extreme denaturing conditionstypically render the template to be amplified substantially singlestranded, posing a challenge for the large number of applicationsinvolving multiplex clonal amplification, i.e., clonal amplification ofmultiple different templates within the same reaction mixture. For suchmultiplex applications, use of these extreme denaturing conditions canbe counterproductive because it typically results in the liberation ofone strand of the template from its associated location, leaving theliberated strand free to migrate within the solution and contaminateother amplicons developing in close proximity. Such cross-contaminationtypically results in reduced yields of monoclonal amplified populationsand increased yield of polyclonal contaminants that typically not usefulfor many downstream applications. There exists a need for improvednucleic acid amplification methods (as well as associated compositions,systems, and kits) that eliminate the pitfalls associated withconventional amplification methods.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems and apparatuses, for nucleic acidamplification, comprising amplifying a nucleic acid template to producean amplicon comprised of a substantially monoclonal population ofpolynucleotides. Monoclonality is typically considered to be desirablein nucleic acid assays because the different characteristics of thediverse polynucleotides within a polyclonal population can complicatethe interpretation of assay data. One example involves nucleic acidsequencing applications, in which the presence of polyclonal populationscan complicate the interpretation of sequencing data; however, with manysequencing systems are not sensitive enough to detect nucleotidesequence data from a single polynucleotide template, thus requiringclonal amplification of templates prior to sequencing.

In some embodiments, the amplification methods of the present disclosurecan be employed to clonally amplify two or more different nucleic acidtemplates, optionally using and within the same reaction mixture, toproduce at least two substantially monoclonal nucleic acid populations.Optionally, at least one of the substantially monoclonal population isformed via amplification of a single polynucleotide template.

Optionally, the two or more different nucleic acid templates areamplified simultaneously and/or in parallel.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems and kits) for nucleic acidsynthesis, comprising: providing at least two double stranded nucleicacid templates in a reaction mixture; and forming at least twosubstantially monoclonal nucleic acid populations by clonally amplifyingthe at least two double stranded nucleic acid templates according to anyof the methods described herein.

In some embodiments, clonally amplifying optionally includes forming areaction mixture. The reaction mixture can include a continuous liquidphase. In some embodiments, the continuous liquid phase includes asingle continuous aqueous phase. The liquid phase can include two ormore polynucleotide templates, which can optionally have the samenucleotide sequence, or can have nucleotide sequences that are differentfrom each other. In some embodiments, at least one of the two or morepolynucleotide templates can include at least one nucleic acid sequencethat is substantially non-identical, or substantially non-complementary,to at least one other polynucleotide template within the reactionmixture.

In some embodiments, the two or more different nucleic acid templatesare localized, deposited or positioned at different sites prior to theamplifying.

In some embodiments, the two or more different nucleic acid templatesare clonally amplified in solution, optionally within a single reactionmixture, and the resulting two or more substantially monoclonal nucleicacid populations are then localized, deposited or positioned atdifferent sites following such clonal amplification.

The different sites are optionally members of an array of sites. Thearray can include a two-dimensional array of sites on a surface (e.g.,of a flowcell, electronic device, transistor chip, reaction chamber,channel, and the like), or a three-dimensional array of sites within amatrix or other medium (e.g., solid, semi-solid, liquid, fluid, and thelike).

Optionally, the two or more different nucleic acid templates areamplified within a continuous liquid phase, typically a continuousaqueous phase, of the same reaction mixture, thereby producing two ormore different and substantially monoclonal populations ofpolynucleotides, each population being generated via amplification of asingle polynucleotide template present in the reaction mixture.

Optionally, the continuous liquid phase is contained within a single orsame phase of the reaction mixture.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems and kits) for nucleic acidsynthesis, comprising: providing a double stranded nucleic acidtemplate; and forming a substantially monoclonal nucleic acid populationby amplifying the double stranded nucleic acid template. Optionally, theamplifying includes clonally amplifying the double stranded nucleic acidtemplate.

Optionally, the amplifying includes performing at least one round ofamplification under substantially isothermal conditions.

Optionally, the amplifying includes performing at least two consecutivecycles of nucleic acid synthesis under substantially isothermalconditions.

In some embodiments, the amplifying includes recombinase polymeraseamplification (RPA). For example, the amplifying can include performingat least one round of RPA.

In some embodiments, the amplifying includes template walking. Forexample, the amplifying can include performing at least one round oftemplate walking.

In some embodiments, the amplifying optionally includes performing twodifferent rounds of amplification within the sites or reaction chambers.For example, the amplifying can include performing at least one round ofRPA within the sites or reaction chambers, and performing at least oneround of template walking within the sites or reaction chambers, in anyorder or combination of rounds. In some embodiments, at least twoconsecutive cycles in any one or more of the rounds of amplification areperformed under substantially isothermal conditions. In someembodiments, at least one of the rounds of amplification is performedunder substantially isothermal conditions.

Optionally, the nucleic acid template to be amplified is doublestranded, or is rendered at least partially double stranded usingappropriate procedures prior to amplification. (The template to beamplified is referred to interchangeably herein as a nucleic acidtemplate or a polynucleotide template). In some embodiments, thetemplate is linear. Alternatively, the template can be circular, orinclude a combination of linear and circular regions.

Optionally, the double stranded nucleic acid template includes a forwardstrand. The double stranded nucleic acid template can further include areverse strand. The forward strand optionally includes a first primerbinding site. The reverse strand optionally includes a second primerbinding site.

In some embodiments, the template already includes a first and/or secondprimer binding site. Alternatively, the template optionally does notoriginally include a primer binding site, and the disclosed methodsoptionally include attaching or introducing a primer binding site to thetemplate prior to the amplifying. For example, the method can optionallyinclude ligating or otherwise introducing an adaptor containing a primerbinding site to, or into, the templates. The adapter can be ligated orotherwise introduced to an end of a linear template, or within the bodyof a linear or circular template. Optionally, the template can becircularized after the adapter is ligated or introduced. In someembodiments, a first adapter can be ligated or introduced at a first endof a linear template, and a second adaptor can be ligated or introducedat a second end of the template.

In some embodiments, the amplifying includes contacting the partiallydenatured template with a first primer, with a second primer, or withboth a first primer and a second primer, in any order or combination.

In some embodiments, the first primer contains a first primer sequence.The first primer optionally includes an extendible end (e.g., a 3′OHcontaining end). The first primer can optionally be attached to acompound (e.g., a “drag tag”), or to a support (e.g., a bead or asurface of the site or reaction chamber).

In some embodiments, the second primer contains a second primersequence. The second primer optionally includes an extendible end (e.g.,a 3′OH containing end). The second primer can optionally be attached toa compound (e.g., a “drag tag”), or to a support (e.g., a bead or asurface of the site or reaction chamber).

Optionally, the first primer binds to the first primer binding site toform a first primer-template duplex. The second primer can bind to thesecond primer binding site to form a second primer-template duplex.

In some embodiments, amplifying includes extending the first primer toform an extended first primer. For example, amplifying can includeextending the first primer of the first primer-template duplex to forman extended first primer.

In some embodiments, amplifying includes extending the first primer toform an extended first primer. For example, amplifying can includeextending the first primer of the first primer-template duplex to forman extended first primer.

Optionally, the extending is performed by a polymerase. The polymerasecan be a strand-displacing polymerase.

In some embodiments, the amplifying includes contacting the template tobe amplified with a recombinase.

In some embodiments, the amplifying includes forming a partiallydenatured template. For example, the amplification can include partiallydenaturing the double stranded nucleic acid template.

Optionally, partially denaturing includes subjecting the double strandednucleic acid template to partially denaturing conditions.

In some embodiments, partially denaturing conditions includetemperatures that are less than the Tm of the nucleic acid templateincluding, for example, at least 5° C., 10° C., 15° C., 20° C., 25° C.or 50° C. below the Tm of the nucleic acid template. In someembodiments, partially denaturing conditions include temperaturesgreater (for example, at least 5° C., 10° C., 15° C., 20° C., 25° C. or50° C. greater) than the Tm of the first primer, the second primer, orboth the first and second primer. In some embodiments, partiallydenaturing conditions include temperatures greater (for example, atleast 5° C., 10° C., 15° C., 20° C., 25° C. or 50° C. greater) than theTm of the first primer binding site, the second primer binding site, orboth the first primer binding site and the second primer binding site.In some embodiments, the nucleic acid template can include an adaptorsequence at one or both ends, and the partially denaturing conditionscan include temperatures greater than the Tm of the adaptor sequence. Insome embodiments, partially denaturing conditions (particularlypartially denaturing temperatures) are employed to selectively amplifynucleic acid templates in a “template walking” process, as describedfurther herein.

In other embodiments, partially denaturing conditions include treatingor contacting the nucleic acid templates to be amplified with one ormore enzymes that are capable of partially denaturing the nucleic acidtemplate, optionally in a sequence-specific or sequence-directed manner.In some embodiments, at least one enzyme catalyzes strand invasionand/or unwinding, optionally in a sequence-specific manner. Optionally,the one or more enzymes include one or more enzymes selected from thegroup consisting of: recombinases, topoisomerases and helicases. In someembodiments, partially denaturing the template can include contactingthe template with a recombinase and forming a nucleoprotein complexincluding the recombinase. Optionally, the template is contacted with arecombinase in the presence of a first primer, a second primer, or botha first and second primer. Partially denaturing can include catalyzingstrand exchange using the recombinase and hybridizing the first primerto the first primer binding site (or hybridizing the second primer tothe second primer binding site). In some embodiments, partiallydenaturing includes performing strand exchange and hybridizing both thefirst primer to the first primer binding site and the second primer tothe second primer binding site using the recombinase.

In some embodiments, the partially denatured template includes a singlestranded portion and a double stranded portion. In some embodiments, thesingle stranded portion includes the first primer binding site. In someembodiments, the single stranded portion includes the second primerbinding site. In some embodiments, the single stranded portion includesboth the first primer binding site and the second primer binding site.

In some embodiments, partially denaturing the template includescontacting the template with one or more nucleoprotein complexes. Atleast one of the nucleoprotein complexes can include a recombinase. Atleast one of the nucleoprotein complexes can include a primer (e.g., afirst primer or a second primer, or a primer including a sequencecomplementary to a corresponding primer binding sequence in thetemplate). In some embodiments, partially denaturing the template caninclude contacting the template with a nucleoprotein complex including aprimer. Partially denaturing can include hybridizing the primer of thenucleoprotein complex to the corresponding primer binding site in thetemplate, thereby forming a primer-template duplex.

In some embodiments, partially denaturing the template can includecontacting the template with a first nucleoprotein complex including afirst primer. Partially denaturing can include hybridizing the firstprimer of the first nucleoprotein complex to the first primer bindingsite of the forward strand, thereby forming a first primer-templateduplex.

In some embodiments, partially denaturing the template can includecontacting the template with a second nucleoprotein complex including asecond primer. Partially denaturing can include hybridizing the secondprimer of the second nucleoprotein complex to the second primer bindingsite of the reverse strand, thereby forming a second primer-templateduplex.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include one or more primer extensionsteps. For example, the methods can include extending a primer vianucleotide incorporation using a polymerase.

In some embodiments, the polymerase is a strand-displacing polymerase.

Optionally, extending a primer includes contacting the primer with apolymerase and one or more types of nucleotides under nucleotideincorporation conditions. In some embodiments, the one or more types ofnucleotides do not include extrinsic labels, particularly opticallydetectable labels, for example fluorescent moieties or dyes. Optionally,the reaction mixture includes nucleotides that are naturally occurringnucleotides. Optionally, the nucleotides do not include groups thatterminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like). Typically, extending a primer occurs in atemplate-dependent fashion.

Optionally, the disclosed methods (and related compositions, systems andkits) include extending the first primer by incorporating one or morenucleotides into the first primer of the first primer-template duplexusing the polymerase, thereby forming an extended first primer.

Optionally, the disclosed methods (and related compositions, systems andkits) include binding a second primer to the second primer binding siteof the first extended primer by any suitable method (e.g., ligation orhybridization).

Optionally, the disclosed methods (and related compositions, systems andkits) include extending the second primer by incorporating one or morenucleotides into the second primer of the second primer-template duplexusing the polymerase, thereby forming an extended second primer.

In some embodiments, extending the first primer results in formation ofa first extended primer. The first extended primer can include some orall of the sequence of the reverse strand of the template. Optionally,the first extended primer includes a second primer binding site.

In some embodiments, extending the second primer results in formation ofa second extended primer. The second extended primer can include some orall of the sequence of the forward strand of the template. Optionally,the second extended primer includes a first primer binding site.

In some embodiments, the methods are performed without subjecting thedouble stranded nucleic acid template to extreme denaturing conditionsduring the amplifying. For example, the methods can be performed withoutsubjecting the nucleic acid template(s) to temperatures equal to orgreater than the Tm of the template(s) during the amplifying. In someembodiments, the methods can be performed without contacting thetemplate(s) with chemical denaturants such as NaOH, urea, guanidium, andthe like, during the amplifying. In some embodiments, the amplifyingincludes isothermally amplifying.

In some embodiments, the methods are performed without subjecting thenucleic acid template(s) to extreme denaturing conditions during atleast two, three, four, or more than four, consecutive cycles of nucleicacid synthesis. For example, the methods can include two, three, four,or more than four, consecutive cycles of nucleic acid synthesis withoutcontacting the nucleic acid template(s) with a chemical denaturant. Insome embodiments, the methods can include performing two, three, four,or more than four, consecutive cycles of nucleic acid synthesis withoutsubjecting the nucleic acid template(s) to temperatures that are greaterthan 25, 20, 15, 10, 5, 2 or 1° C. below the actual or calculated Tm ofthe template, or population of templates (or the actual or calculatedaverage Tm of the template, or population of templates). The two, three,four, or more than four, consecutive cycles of nucleic acid synthesismay include intervening steps of partial denaturation and/or primerextension.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include linking one or more extendedprimer strands to a support. The linking can optionally be performedduring the amplifying, or alternatively after the amplification iscomplete. In some embodiments, the support includes multiple instancesof a second primer, and the methods can include hybridizing at least oneof the extended first primer strands to a second primer of the support.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include linking one or more extendedsecond primer strands to a support. In some embodiments, the support isattached to a first primer. For example, the support can includemultiple instances of a first primer, and the methods can includehybridizing at least one of the extended second primers to a firstprimer of the support, thereby linking the extended second primer to thesupport. For example, the first primer can hybridize to a first primerbinding site in the extended second primer.

In some embodiments, the support is attached to a second primer. Forexample, the support can include multiple instances of a second primer,and the methods can include hybridizing at least one of the extendedfirst primers to a second primer of the support, thereby linking theextended first primer to the support. For example, the first primer canhybridize to a second primer binding site in the extended first primer.

In some embodiments, the support includes both at least one first primerand at least one second primer, and the disclosed methods (and relatedcompositions, systems and kits) including linking both an extended firstprimer and an extended second primer to the support.

Optionally, the support is attached to a target-specific primer. Thetarget-specific primer optionally hybridizes (or is capable ofhybridizing) to a first subset of templates within the reaction mixture,but is unable to bind to a second subset of templates within thereaction mixture.

Optionally, the support is attached to a universal primer. The universalprimer optionally hybridizes (or is capable of hybridizing) to all, orsubstantially all, of the templates within the reaction mixture.

Optionally, the reaction mixture includes a first support covalentlyattached to a first target-specific primer and a second supportcovalently attached to a second target-specific primer, and wherein thefirst and second target-specific primers are different from each other.

Optionally, the first target-specific primer is substantiallycomplementary to a first target nucleic acid sequence and the secondtarget-specific primer is substantially complementary to a second targetnucleic acid sequence, and wherein the first and second target nucleicacid sequences are different.

In some embodiments, the disclosed methods include forming a firstamplicon by amplifying a first template onto a first support, andforming a second amplicon by amplifying a second template onto a secondsupport, optionally within the same continuous phase of a reactionmixture. The first amplicon is optionally linked or attached to thefirst support, and the second amplicon is optionally linked or attachedto the second support.

The disclosed methods optionally comprise producing two or moremonoclonal, or substantially monoclonal, amplicons by clonallyamplifying two or more polynucleotide templates. The two or morepolynucleotide templates are optionally clonally amplified within acontinuous liquid phase of an amplification reaction mixture. Thecontinuous liquid phase of the amplification reaction mixture caninclude a continuous aqueous phase. In some embodiments, the amplifyingincludes generating at least two substantially monoclonal populations ofamplified polynucleotides, each of said populations being formed viaamplification of a single polynucleotide template. Optionally, theclonally amplifying includes at least one round of RPA. Optionally, theclonally amplifying includes at least one round of template walking.

In some embodiments, the amplifying optionally includes forming anamplification reaction mixture including a continuous liquid phase. Insome embodiments, the continuous liquid phase is a single continuousaqueous phase. The liquid phase can include two or more polynucleotidetemplates, which can optionally be different from each other. Forexample, the two or more polynucleotide templates can include at leastone nucleic acid sequence that is substantially non-identical, orsubstantially non-complementary, to at least one other polynucleotidetemplate within the amplification reaction mixture.

In some embodiments, the amplifying optionally includes forming anamplification reaction mixture including a single continuous aqueousphase having two or more polynucleotide templates. Amplifying optionallyincludes forming two or more substantially monoclonal nucleic acidpopulations by clonally amplifying the two or more polynucleotidetemplates within the single aqueous phase. Optionally, the clonallyamplifying includes at least one round of RPA. Optionally, the clonallyamplifying includes at least one round of template walking.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems and kits) for amplifying one or morenucleic acid templates, optionally in parallel, using partiallydenaturing conditions. In some embodiments, two or more templates areamplified using such methods, optionally in array format. Optionally,the templates are amplified in bulk in solution prior to distributioninto the array. Alternatively, the templates are first distributed tosites in the array and then amplified in situ at (or within) the sitesof the array.

Optionally, the template is single stranded or double stranded. Thetemplate optionally includes one or more primer binding sites.

In some embodiments, the methods can include subjecting adouble-stranded nucleic acid template including a primer binding site onat least one strand to at least one cycle of template-based replicationusing a polymerase.

Optionally, the at least one cycle of template-based replicationincludes a partial denaturation step, an annealing step, and anextension step.

In some embodiments, the methods include amplifying the double strandednucleic acid template by subjecting the template to at least twoconsecutive cycles of template-based replication.

In some embodiments, the methods include partially denaturing thetemplate. Optionally, the methods include forming a partially denaturedtemplate including a single stranded region. The partially denaturedtemplate can also include a double stranded region. The single strandedregion can contain the primer binding site.

Optionally, the partially denaturing includes subjecting the template toa temperature that is at least 20, 15, 10, 5, 2, or 1° C. lower than theTm of the primer binding site.

Optionally, the partially denaturing includes subjecting the template toa temperature that is equal to or greater than the Tm of the primerbinding site.

Optionally, the partially denaturing includes contacting the doublestranded template with a recombinase and a primer. The recombinase andprimer may form part of a nucleoprotein complex, and the partiallydenaturing includes contacting the template with the complex.

In some embodiments, the methods include forming a primer-templateduplex by hybridizing a primer to the primer binding site of the singlestranded region. In some embodiments, the primed template includes adouble stranded region. Optionally, the double stranded region does notcontain a primer binding site.

In some embodiments, the methods include extending the primer of theprimer-template duplex. Optionally, the methods include forming anextended primer.

In some embodiments, different templates can be clonally amplified ontodifferent discrete supports (e.g., beads or particles) without the needfor compartmentalization prior to amplification. In other embodiments,the templates are partitioned or distributed into emulsions prior toamplifying. Optionally, the templates are distributed into dropletsforming part of a hydrophilic phase of an emulsion having adiscontinuous hydrophilic phase and a continuous hydrophobic phase. Insome embodiments, the emulsion droplets of the hydrophilic phase alsoinclude one or more components necessary to practice RPA. For example,the emulsion droplets can include a recombinase. Optionally, thedroplets include a strand-displacing polymerase. In some embodiments,the droplets include a support-immobilized primer and/or a solutionphase primer. Optionally, the primer can bind to the template, or to anamplification product thereof.

In some embodiments, the disclosed compositions, systems, methods,apparatuses and kits for nucleic acid amplification using emulsion-basedamplification comprising nucleic acid synthesis following partialdenaturation of the template offer advantages over conventionalamplification methods (including emulsion based PCR or emPCR involvingtraditional thermocycling). For example, a nucleic acid amplificationreaction comprising emulsion-based RPA (“emRPA”), or emulsion-basedtemplate walking, can yield longer amplified polynucleotide templates,fewer amplification steps, reduced time for preparing amplifiedpolynucleotide templates, and/or increased quality of sequencing data.Some suitable emulsion compositions for use with the disclosedamplification methods can be found, for example, in U.S. Pat. Nos.7,622,280, 7,601,499 and 7,323,305, incorporated by reference herein intheir entireties.

In some embodiments, the methods include providing a double strandedtemplate including a forward strand containing a first primer bindingsite, a reverse strand containing a second primer binding site;partially denaturing the template and forming a partially denaturedtemplate including a single stranded region containing the first primerbinding site and at least one double stranded region; forming a firstprimer-template duplex by hybridizing a first primer to the first primerbinding site of the single stranded region; extending the first primerof the first primer-template duplex using a polymerase to form anextended first primer including a second primer binding site, whereinthe extended first primer is at least partially hybridized to theforward strand of the template; partially denaturing the extended firststrand from the template to form a single stranded region including asecond primer binding site; hybridizing a second primer to the secondprimer binding site of the single stranded region and forming a secondprimer-template duplex, and extending the second primer of the secondprimer-template duplex, thereby forming an extended second primer.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems and kits) for a method for nucleic acidsynthesis from a nucleic acid template, comprising: providing a firstnucleic acid duplex including a forward strand and a reverse strand,wherein the forward strand contains a forward primer binding site andthe reverse strand contains a reverse primer binding site and whereinthe first duplex has a first melting temperature (“template Tm”), theforward primer binding site has a second melting temperature (“forwardprimer Tm”) and the reverse primer binding site has a third meltingtemperature (“reverse primer Tm”); partially denaturing the firstduplex, wherein the partially denatured first duplex includes asingle-stranded region containing the forward primer binding site and atleast one double stranded region; forming a primed first duplex byhybridizing a forward primer to the forward primer binding site of thepartially denatured first duplex; extending the forward primer bycontacting the primed first duplex with a strand displacing polymeraseand nucleotides under primer extension condition, thereby forming secondduplex having a fourth melting temperature (“fourth Tm”) and includingone strand containing the forward primer binding site and one strandcontaining the reverse primer binding site; partially denaturing thesecond duplex, wherein the partially denatured second duplex includes asingle-stranded region containing the reverse primer binding site and atleast one double stranded region; forming a reverse primed second duplexby hybridizing a reverse primer to the reverse primer binding site ofthe partially denatured second duplex; extending the reverse primer ofthe reverse primed second duplex by contacting the reverse primed secondduplex with a strand displacing polymerase and nucleotides under primerextension conditions.

In some embodiments, the methods (and related compositions, systems, andkits) can further include sequencing an amplified template, orsequencing an extended primer, (e.g. an extended first primer, orextended second primer). The sequencing can include any suitable methodof sequencing known in the art. In some embodiments, the sequencingincludes sequencing by synthesis or sequencing by electronic detection(e.g., nanopore sequencing). In some embodiments, sequence includesextending a template or amplified template, or extending a sequencingprimer hybridized to a template or amplified template, via nucleotideincorporation by a polymerase. In some embodiments, sequencing includessequence a template or amplified template that is attached to a supportby contacting the template or extended primer with a sequencing primer,a polymerase, and at least one type of nucleotide. In some embodiments,the sequencing includes contacting the template, or amplified template,or extended primer, with a sequencing primer, a polymerase and with onlyone type of nucleotide that does not include an extrinsic label or achain terminating group.

Optionally, the template (or amplified product) can be deposited,localized, or positioned, to a site. In some embodiments, multipletemplates/amplified templates/extended first primers are deposited orpositioned to different sites in an array of sites. In some embodiments,the depositing, positioning or localizing is performed prior toamplification of the template. In some embodiments, the depositing,positioning or localizing is performed after the amplifying. Forexample, amplified templates or extended first primers can be deposited,positioned or localized to different sites of an array.

The disclosed methods result in the production of a plurality ofamplicons, at least some of which amplicons include a clonally amplifiednucleic acid population. The clonally amplified populations produced bythe methods of the disclosure can be useful for a variety of purposes.In some embodiments, the disclosed methods (and related compositions,systems and kits) optionally include further analysis and/ormanipulation of the clonally amplified populations (amplicons). Forexample, in some embodiments, the numbers of amplicons exhibitingcertain desired characteristics can be detected and optionallyquantified.

In some embodiments, the methods can include determining which of thediscrete supports (e.g., beads), include amplicons. Similarly, themethods can include determining which sites of an array that includeamplicons. The presence of amplicons at supports or sites can optionallybe detected determined using DNA-based detection procedures such as UVabsorbance, staining with DNA-specific dyes, TAQMAN® assays, qPCR,hybridization to fluorescent probes, and the like. In some embodiments,the methods can include determining which bead supports (or sites of anarray) have received substantially monoclonal amplicons. For example,the bead supports (or array sites) can be analyzed to determine whichsupports or sites can produce a detectable and coherent (i.e.,analyzable) sequence-dependent signal.

In some embodiments, the amplifying is followed by sequencing theamplified product. The amplified product that is sequenced can includean amplicon comprising a substantially monoclonal nucleic acidpopulation. In some embodiments, the disclosed methods include formingor positioning single members of a plurality of amplicons to differentsites. The different sites optionally form part of an array of sites. Insome embodiments, the sites in the array of sites include wells(reaction chambers) on the surface of an isFET array, as describedfurther herein.

In some embodiments, the methods (and related compositions, systems, andkits) can further include sequencing an amplified template, orsequencing an extended primer, (e.g. an extended first primer, orextended second primer). The sequencing can include any suitable methodof sequencing known in the art. In some embodiments, the sequencingincludes sequencing by synthesis or sequencing by electronic detection(e.g., nanopore sequencing). In some embodiments, sequence includesextending a template or amplified template, or extending a sequencingprimer hybridized to a template or amplified template, via nucleotideincorporation by a polymerase. In some embodiments, sequencing includessequence a template or amplified template that is attached to a supportby contacting the template or extended primer with a sequencing primer,a polymerase, and at least one type of nucleotide. In some embodiments,the sequencing includes contacting the template, or amplified template,or extended primer, with a sequencing primer, a polymerase and with onlyone type of nucleotide that does not include an extrinsic label or achain terminating group.

In some embodiments, methods of downstream analysis include sequencingat least some of the plurality of amplicons in parallel. Optionally, themultiple templates/amplified templates/extended first primers situatedat different sites of the array are sequenced in parallel.

In some embodiments, the sequencing can include binding a sequencingprimer to the nucleic acids of at least two different amplicons, or atleast two different substantially monoclonal populations.

In some embodiments, the sequencing can include incorporating anucleotide into the sequencing primer using the polymerase. Optionally,the incorporating includes forming at least one nucleotide incorporationbyproduct.

Optionally, the nucleic acid to be sequenced is positioned at a site.The site can include a reaction chamber or well. The site can be part ofan array of similar or identical sites. The array can include atwo-dimensional array of sites on a surface (e.g., of a flowcell,electronic device, transistor chip, reaction chamber, channel, and thelike), or a three-dimensional array of sites within a matrix or othermedium (e.g., solid, semi-solid, liquid, fluid, and the like).

In some embodiments, the site is operatively coupled to a sensor. Themethod can include detecting the nucleotide incorporation using thesensor. Optionally, the site and the sensor are located in an array ofsites coupled to sensors.

In some embodiments, the site comprises a hydrophilic polymer matrixconformally disposed within a well operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix includes a hydrogel polymermatrix.

Optionally, the hydrophilic polymer matrix is a cured-in-place polymermatrix.

Optionally, the hydrophilic polymer matrix includes polyacrylamide,copolymers thereof, derivatives thereof, or combinations thereof.

Optionally, the polyacrylamide is conjugated with an oligonucleotideprimer.

Optionally, the well has a characteristic diameter in a range of 0.1micrometers to 2 micrometers.

Optionally, the well has a depth in a range of 0.01 micrometers to 10micrometers.

In some embodiments, the sensor includes a field effect transistor(FET). The FET can include an ion sensitive FET (ISFET).

In some embodiments, the methods (and related compositions, systems andkits) can include detecting the presence of one or more nucleotideincorporation byproducts at a site of the array, optionally using theFET.

In some embodiments, the methods can include detecting a pH changeoccurring within the at least one reaction chamber, optionally using theFET.

In some embodiments, the disclosed methods include introducing anucleotide into the site; and detecting an output signal from the sensorresulting from incorporation of the nucleotide into the sequencingprimer. The output signal is optionally based on a threshold voltage ofthe FET. In some embodiments, the FET includes a floating gate conductorcoupled to the site.

In some embodiments, the FET includes a floating gate structurecomprising a plurality of conductors electrically coupled to one anotherand separated by dielectric layers, and the floating gate conductor isan uppermost conductor in the plurality of conductors.

In some embodiments, the floating gate conductor includes an uppersurface defining a bottom surface of the site.

In some embodiments, the floating gate conductor comprises anelectrically conductive material, and the upper surface of the floatinggate conductor includes an oxide of the electrically conductivematerial.

In some embodiments, the floating gate conductor is coupled to the atleast one reaction chamber via a sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the amplification reaction mixture can include arecombinase. The recombinase can include any suitable agent that canpromote recombination between polynucleotide molecules. The recombinasecan be an enzyme that catalyzes homologous recombination. For example,the amplification reaction mixture can include a recombinase thatincludes, or is derived from, a bacterial, eukaryotic or viral (e.g.,phage) recombinase enzyme.

In some embodiments, the amplification reaction mixture includes anenzyme that can bind a primer and a polynucleotide template to form acomplex, or can catalyze strand invasion of the polynucleotide templateto form a D-loop structure. In some embodiments, the amplificationreaction mixture includes one or more proteins selected from the groupconsisting of: UvsX, RecA and Rad51.

In some embodiments, the amplification reaction mixture can include arecombinase accessory protein, for example UvsY.

In some embodiments, the amplification reaction mixture can include asingle stranded binding protein (SSBP).

In some embodiments, the amplification reaction mixture can include apolymerase. The polymerase optionally has, or lacks, exonucleaseactivity. In some embodiments, the polymerase has 5′ to 3′ exonucleaseactivity, 3′ to 5′ exonuclease activity, or both. Optionally, thepolymerase lacks any one or more of such exonuclease activities.

In some embodiments, the polymerase has strand displacing activity.

In some embodiments, the amplification reaction mixture can include oneor more solid or semi-solid supports. At least one of the supports caninclude one or more instances of a first primer including a first primersequence. In some embodiments, at least one polynucleotide template inthe reaction mixture includes a first primer binding sequence. The firstprimer binding sequence can be substantially identical or substantiallycomplementary to the first primer sequence. In some embodiments, atleast one, some or all of the supports include a plurality of firstprimers that are substantially identical to each other. In someembodiments, all of the primers on the supports are substantiallyidentical to each other, or all include a substantially identical firstprimer sequence.

In some embodiments, at least one of the supports includes two or moredifferent primers attached thereto. For example, the at least onesupport can include at least one instance of the first primer and atleast one instance of a second primer.

In some embodiments, the aqueous phase of the reaction mixture includesa plurality of supports, at least two supports of the plurality beingattached to primers including a first priming sequence. In someembodiments, the reaction mixture includes two or more differentpolynucleotide templates having a first primer binding sequence.

In some embodiments, the amplification reaction mixture can include adiffusion limiting agent. The diffusion limiting agent can be any agentthat is effective in preventing or slowing the diffusion of one or moreof the polynucleotide templates and/or one or more of the amplificationreaction products through the amplification reaction mixture.

In some embodiments, the amplification reaction mixture can include asieving agent. The sieving agent can be any agent that is effective insieving one or more polynucleotides present in the amplificationreaction mixture, such as for example amplification reaction productsand/or polynucleotide templates. In some embodiments, the sieving agentrestricts or slows the migration of polynucleotide amplificationproductions through the reaction mixture.

In some embodiments, the amplification reaction mixture can include acrowding agent.

In some embodiments, the amplification reaction mixture includes both acrowding agent and a sieving agent.

In some embodiments, the disclosed methods comprise clonally amplifyingat least two of the two or more polynucleotide templates by (a) formingan amplification reaction mixture including a single continuous liquidphase containing two or more polynucleotide templates, one or moresurfaces or supports, and amplification components; and (b) clonallyamplifying at least two of said polynucleotide templates onto one ormore supports. Optionally, the clonally amplifying includes forming atleast two different substantially monoclonal amplicons. In someembodiments, the clonally amplifying includes subjecting theamplification reaction mixture to amplification conditions. In someembodiments, two or more of said amplicons are each linked to a surfaceor support. For example, the amplification reaction mixture may includea single support or surface, such that each polynucleotide templateattaches to a given region of the support or surface.

In some embodiments, methods for nucleic acid amplification can beconducted in a single reaction vessel.

In some embodiments, methods for nucleic acid amplification can beconducted in a single continuous liquid phase that does not providecompartmentalization of the multiple nucleic acid amplificationreactions occurring in a single reaction vessel. In some embodiments,methods for nucleic acid amplification can be conducted in water-in-oilemulsions that provide compartmentalization (micro-reactors).

In some embodiments, the methods for nucleic acid amplification can bepracticed to attach a plurality of polynucleotides to a support orsurface. For example, the methods can comprise forming a reactionmixture containing at least one surface, and subjecting the reactionmixture to amplification conditions. In some embodiments, a surfaceincludes the surface of a bead, a planar surface or an interior wall ofa channel or tube.

In some embodiments, methods for nucleic acid amplification comprise:(a) forming an amplification reaction mixture including a singlecontinuous liquid phase containing a plurality of beads, a plurality ofdifferent polynucleotides and a recombinase; (b) subjecting theamplification reaction mixture to isothermal amplification conditions,thereby generating a plurality of beads attached to substantiallymonoclonal nucleic acid populations attached thereto.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems and kits) for array-basedamplification of nucleic acid templates directly onto the surface of anarray, resulting in the formation of any array whose individual featuresinclude amplicons containing substantially monoclonal populations ofamplified product. These embodiments contrast with other embodimentsdescribed herein where the nucleic acid templates are optionallyamplified in solution onto discrete supports (e.g., beads), which arethen distributed into an array.

In some embodiments, methods (as well as related compositions, systemsand kits) for array-based amplification are provided. In someembodiments, different polynucleotide templates are distributed into anarray of sites and then amplified in situ. The resulting array ofamplicons can then be analyzed using appropriate downstream procedures.

In some embodiments, methods for nucleic acid amplification comprise:

a) distributing at least two different polynucleotides into sites byintroducing a single one of said polynucleotides into at least two ofsaid sites that are in fluid communication with each other; and (b)forming at least two substantially monoclonal nucleic acid populationsby amplifying the polynucleotides within said at least two sites. Thesites can optionally include surfaces, wells, grooves, flowcells,reaction chambers or channels. In some embodiments, the amplifying isperformed without sealing the sites from each other. For example, the atleast two sites can remain in fluid communication with each other duringthe amplifying.

In some embodiments, methods for nucleic acid amplification comprise:

a) distributing at least two different polynucleotides into sites byintroducing a single one of said polynucleotides into at least two ofsaid sites; and (b) forming at least two substantially monoclonalnucleic acid populations by amplifying the polynucleotides within saidat least two sites. The sites can optionally include surfaces, wells,grooves, flowcells, reaction chambers or channels. In some embodiments,the amplifying is performed without sealing the sites from each other.For example, the at least two sites can remain in fluid communicationwith each other during the amplifying.

In some embodiments, the sites comprise reaction chambers, and themethods for nucleic acid amplification comprise: (a) distributing atleast two polynucleotide templates into reaction chambers by introducinga single one of said polynucleotides into at least two of said reactionchambers that are in fluid communication with each other; and (b)forming at least two substantially monoclonal nucleic acid populationsby amplifying the polynucleotide templates within said at least tworeaction chambers. In some embodiments, the amplifying is performedwithout sealing the reaction chambers from each other. For example, theat least two reaction chambers can remain in fluid communication witheach other during the amplifying.

In some embodiments, the sites comprise reaction chambers, and themethods for nucleic acid amplification comprise: (a) distributing atleast two different polynucleotides into reaction chambers byintroducing a single one of said polynucleotides into at least two ofsaid reaction chambers that are in fluid communication with each other;and (b) forming at least two substantially monoclonal nucleic acidpopulations by amplifying the polynucleotides within said at least tworeaction chambers. In some embodiments, the amplifying is performedwithout sealing the reaction chambers from each other. For example, theat least two reaction chambers can remain in fluid communication witheach other during the amplifying.

In some embodiments, the amplifying step of any and all methods of thedisclosure can be performed without completely denaturing thepolynucleotides during the amplifying. For example, the disclosedmethods can include amplifying the at least two differentpolynucleotides via isothermal amplification. The amplifying can includeamplifying the at least two different polynucleotides undersubstantially isothermal conditions. Optionally, the amplifying isperformed without contacting the polynucleotides with chemicaldenaturants during the amplifying.

Optionally, the amplifying includes performing at least one round ofamplification under substantially isothermal conditions.

Optionally, the amplifying includes performing at least two consecutivecycles of nucleic acid synthesis under substantially isothermalconditions.

In some embodiments, the amplifying includes recombinase polymeraseamplification (RPA). For example, the amplifying can include performingat least one round of RPA.

In some embodiments, the amplifying includes template walking. Forexample, the amplifying can include performing at least one round oftemplate walking.

In some embodiments, the amplifying optionally includes performing twodifferent rounds of amplification within the sites or reaction chambers.For example, the amplifying can include performing at least one round ofRPA within the sites or reaction chambers, and performing at least oneround of template walking within the sites or reaction chambers, in anyorder or combination of rounds. In some embodiments, at least twoconsecutive cycles in any one or more of the rounds of amplification areperformed under substantially isothermal conditions. In someembodiments, at least one of the rounds of amplification is performedunder substantially isothermal conditions.

In some embodiments, amplifying includes contacting the polynucleotidesto be amplified with a reaction mixture. The contacting can optionallybe performed prior to or after the distributing; it is to be understoodthat the present disclosure embraces embodiments where thepolynucleotides are contacted with individual components (orcombinations of components) of the reaction mixture at different timesand in series, as well as embodiments where are any one or somecomponents of the reaction mixture are contacted with the at least twodifferent polynucleotides prior to the distributing, while the remainingcomponents of the reaction mixture are contacted with the at least twodifferent polynucleotides after the distributing.

The at least two polynucleotides that are distributed can optionallyserve as templates for nucleic acid synthesis within their respectivereaction chambers. In some embodiments, the at least two polynucleotidesinclude different sequences. In some embodiments, the polynucleotidesare double stranded or as single stranded prior to distributing. In someembodiments, the polynucleotides are linear, circular, or a combinationof both. In a typical embodiment, the polynucleotides are at leastpartially double stranded (or are distributed in single stranded formand then rendered at least partially double stranded within the sites orreaction chambers after the distributing. The polynucleotides can berendered double stranded prior to the amplifying (especially inembodiments where the amplifying includes RPA or template walking).

In some embodiments, the at least two different polynucleotide templatesto be amplified each contain a primer binding site, and the amplifyingincludes binding a primer to the primer binding site to form aprimer-template duplex.

Optionally, the amplifying includes extending the primer of theprimer-template duplex. The extending optionally occurs at or within thesite or reaction chamber of the array. Optionally, extending a primerincludes contacting the primer with a polymerase and one or more typesof nucleotides under nucleotide incorporation conditions. In someembodiments, the one or more types of nucleotides do not includeextrinsic labels, particularly optically detectable labels, for examplefluorescent moieties or dyes. Optionally, the reaction mixture includesnucleotides that are naturally occurring nucleotides. Optionally, thenucleotides do not include groups that terminate nucleic acid synthesis(e.g., dideoxy groups, reversible terminators, and the like). Typically,extending a primer occurs in a template-dependent fashion.

Optionally, the at least two different polynucleotides (i.e., thetemplates to be amplified) individually include a first sequence(referred to as “first primer binding site”) that is substantiallyidentical, or substantially complementary, to at least some portion ofthe first primer.

In some embodiments, the reaction mixture includes a first primercontaining a first primer sequence. The first primer optionally includesan extendible end (e.g., a 3′OH containing end). The first primer canoptionally be attached to a compound (e.g., a “drag tag”), or to asupport (e.g., a bead or a surface of the site or reaction chamber).

Optionally, the disclosed methods (and related compositions, systems andkits) include extending the first primer by incorporating one or morenucleotides into the first primer of the first primer-template duplexusing the polymerase, thereby forming an extended first primer.

In some embodiments, the at least two different polynucleotides includea second sequence (referred to as “second primer binding site”) that issubstantially identical, or substantially complementary, to at leastsome portion of a second primer.

In some embodiments, extending the first primer results in formation ofa first extended primer. The first extended primer can include some orall of the sequence of the reverse strand of the template. Optionally,the first extended primer includes a second primer binding site.

Optionally, the disclosed methods (and related compositions, systems andkits) include binding a second primer to the second primer binding siteof the first extended primer by any suitable method (e.g., ligation orhybridization).

In some embodiments, the second primer contains a second primersequence. The second primer optionally includes an extendible end (e.g.,a 3′OH containing end). The second primer can optionally be attached toa compound (e.g., a “drag tag”), or to a support (e.g., a bead or asurface of the site or reaction chamber).

In some embodiments, the methods include extending the second primer byincorporating one or more nucleotides into the second primer of thesecond primer-template duplex using the polymerase, thereby forming anextended second primer.

In some embodiments, extending the second primer results in formation ofa second extended primer. The second extended primer can include some orall of the sequence of the forward strand of the template. Optionally,the second extended primer includes a first primer binding site.

In some embodiments, amplification is performed using at least oneprimer, typically two or more primers. The at least one primeroptionally comprises one or more modified groups. Optionally, at leastone of the one or more modified groups includes a modified base, amodified sugar or a modified phosphate moiety. In some embodiments, asurface-immobilized primer, or a solution-phase primer, or both, caninclude one or more of the modified groups.

Optionally, the modified group can be located at the terminal 5′ or 3′end, or at any internal location in the primer.

Optionally, the modified group can reduce nucleic acid duplex formation.For example, a primer with a modified group may exhibit reduce ratesand/or levels of nucleic acid duplex formation relative to itsunmodified but otherwise substantially identical counterpart.

Optionally, the modified group can confer exonuclease-resistance to theprimer.

Optionally, the modified group can include a chain terminatingnucleotide.

Optionally, the modified group includes a universal base.

Optionally, the primer includes at least one mismatched base.

Optionally, the primer includes a degenerate sequence.

Optionally, the primer includes a randomized sequence.

Optionally, the primer includes a DNA/RNA hybrid.

Optionally, the modified group can reduce binding between the primer anda recombinase. In some embodiments, the primer including the modifiedgroup is bound at lower rates and/or with a lower binding affinity bythe recombinase than its corresponding unmodified but otherwisesubstantially identical counterpart. In some embodiments, the at leastone primer is bound by a polymerase at rates and/or affinitiescomparable or greater than its corresponding unmodified but otherwisesubstantially identical counterpart.

In some embodiments, the disclosed amplification methods can beperformed using two or more primers, of which at least one primer iscompetent for hybridization and extension of when hybridized to asingle-stranded DNA template. In some embodiments, the at least oneprimer is incompetent as a recombinase substrate.

Optionally, the modified group can include one or more of aribonucleotide, a biotinylated nucleotide, a PEGylated nucleotide, adideoxynucleotide, a 2′-deoxyinosine (hypoxanthine deoxynucleotide), anitroazole, a hydrophobic aromatic non-hydrogen-bonding bases, aphosphorothioate nucleotide, mismatched bases, 2-aminopurine,deoxyuridine, 2′ deoxyinosine, 5-nitroindole, 2′-O-methyl RNA bases,iso-cytosine base, iso-guanosine base, a fluorophore, or an antibody ora epitope-binding fragment thereof.

Optionally, the modified group includes a nucleic acid tail located atthe 5′ end of the primer that can form a hairpin structure.

In some embodiments, the reaction mixture includes one or more primers.For example, the reaction mixture can include at least a firstoligonucleotide primer. In some embodiments, a first primer includes aforward amplification primer which hybridizes to at least a portion ofone strand of a polynucleotide. In some embodiments, a first primercomprises an extendible 3′ end capable of receiving an incomingnucleotide during nucleotide polymerization. In some embodiments,methods for nucleic acid amplification include hybridization to thetemplate of additional oligonucleotide primers (e.g., a second, third,fourth primer, etc.). For example, a second primer can be a reverseamplification primer which hybridizes to at least a portion of onestrand of a double stranded polynucleotide template. In someembodiments, a second primer includes an extendible 3′ end amenable tonucleotide polymerization. In some embodiments, a second primer isattached to a surface. In some embodiments, the second primer has thesame sequence as the first primer. In some embodiments, the secondprimer has a different sequence than the first primer. In someembodiments, a second primer is attached to the same surface as thefirst primer. In this manner, at least a portion of one strand of apolynucleotide can be hybridized to a first primer that is attached to asurface, while at least a different portion of the same polynucleotide(either the same strand or a complementary strand) can be hybridized tothe second primer that is attached to the second surface. In someembodiments, one or both of a first primer or a second primer contains acleavable site. In some embodiments, the cleavable site is selective, inthat one or more specific reagents and or conditions are required toeffect cleavage. For the selectively cleavable site can include adisulfide group, and ester group, an amide group, a thiophosphate ester,an acid anhydride, a 1,2-diol group, a photolytically cleavable group,or other suitable functional groups known in the art. In someembodiments, the cleavable site is cleavable by a sequence-specificendonuclease, such as a restriction endonuclease, many examples of whichare known in the art. In some embodiments, the first primer and secondprimer are attached to the same substrate, and only one of the twoprimers is selectively cleavable. In these embodiments, selectivecleavage of the susceptible primer will release the correspondingcomplementary portion of the polynucleotide from the surface, while theuncleaved primer will remain attached to the surface and hybridized tothe polynucleotide, thereby maintaining attachment of the polynucleotideto the surface. In some embodiments, selective cleavage of thesusceptible primer is performed before, during, or after amplificationof the hybridized polynucleotide.

In some embodiments, the methods are performed without subjecting thedouble stranded nucleic acid template to extreme denaturing conditionsduring the amplifying. For example, the methods can be performed withoutsubjecting the nucleic acid template(s) to temperatures equal to orgreater than the Tm of the template(s) during the amplifying. In someembodiments, the methods can be performed without contacting thetemplate(s) with chemical denaturants such as NaOH, urea, guanidium, andthe like, during the amplifying. In some embodiments, the amplifyingincludes isothermally amplifying.

In some embodiments, the methods are performed without subjecting thenucleic acid template(s) to extreme denaturing conditions during atleast two, three, four, or more than four, consecutive cycles of nucleicacid synthesis. For example, the methods can include two, three, four,or more than four, consecutive steps of nucleic acid synthesis withoutcontacting the nucleic acid template(s) with a chemical denaturant. Insome embodiments, the methods can include performing two, three, four,or more than four, consecutive cycles of nucleic acid synthesis withoutsubjecting the nucleic acid template(s) to temperatures that are greaterthan 25, 20, 15, 10, 5, 2 or 1° C. below the actual or calculated Tm ofthe template, or population of templates (or the actual or calculatedaverage Tm of the template, or population of templates). The two, three,four, or more than four, consecutive cycles of nucleic acid synthesismay include intervening steps of partial denaturation and/or primerextension.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include linking one or more extendedprimer strands to a support. The linking can optionally be performedduring the amplifying, or alternatively after the amplification iscomplete. In some embodiments, the support includes multiple instancesof a second primer, and the methods can include hybridizing at least oneof the extended first primer strands to a second primer of the support.

In some embodiments, the disclosed methods (and related compositions,systems and kits) can further include linking one or more extendedsecond primer strands to a support. In some embodiments, the support isattached to a first primer. For example, the support can includemultiple instances of a first primer, and the methods can includehybridizing at least one of the extended second primers to a firstprimer of the support, thereby linking the extended second primer to thesupport. For example, the first primer can hybridize to a first primerbinding site in the extended second primer. The support can include, forexample, the surface of any array.

In some embodiments, the support is attached to a second primer. Forexample, the support can include multiple instances of a second primer,and the methods can include hybridizing at least one of the extendedfirst primers to a second primer of the support, thereby linking theextended first primer to the support. For example, the first primer canhybridize to a second primer binding site in the extended first primer.

In some embodiments, the support includes both at least one first primerand at least one second primer, and the disclosed methods (and relatedcompositions, systems and kits) including linking both an extended firstprimer and an extended second primer to the support.

Optionally, the support is attached to a target-specific primer. Thetarget-specific primer optionally hybridizes (or is capable ofhybridizing) to a first subset of templates within the reaction mixture,but is unable to bind to a second subset of templates within thereaction mixture.

Optionally, the support is attached to a universal primer. The universalprimer optionally hybridizes (or is capable of hybridizing) to all, orsubstantially all, of the templates within the reaction mixture.

Optionally, the first target-specific primer is substantiallycomplementary to a first target nucleic acid sequence and the secondtarget-specific primer is substantially complementary to a second targetnucleic acid sequence, and wherein the first and second target nucleicacid sequences are different.

In some embodiments, the disclosed methods include forming a firstamplicon by amplifying a first template onto a first support, andforming a second amplicon by amplifying a second template onto a secondsupport, optionally within the same continuous phase of a reactionmixture and at different sites of a surface (e.g., within an array). Thefirst amplicon is optionally linked or attached to the first support,and the second amplicon is optionally linked or attached to the secondsupport.

The disclosed methods optionally comprise producing two or moremonoclonal, or substantially monoclonal, amplicons by clonallyamplifying two or more polynucleotide templates at two or more differentsites of an array of sites, such that at least two sites are formed eachincluding a substantially monoclonal nucleic acid population. The two ormore polynucleotide templates are optionally deposited or positioned atthe different sites and then clonally amplified within a continuousliquid phase of an amplification reaction mixture that is contacted withthe array. The continuous liquid phase of the amplification reactionmixture can include a continuous aqueous phase.

In some embodiments, the amplifying includes generating at least twosubstantially monoclonal populations of amplified polynucleotides, eachof said populations being formed via amplification of a singlepolynucleotide template.

Optionally, the clonally amplifying includes at least one round of RPA.

Optionally, the clonally amplifying includes at least one round oftemplate walking.

In some embodiments, the amplifying optionally includes forming anamplification reaction mixture including a continuous liquid phase. Insome embodiments, the continuous liquid phase is a single continuousaqueous phase. The liquid phase can include two or more polynucleotidetemplates, which can optionally be different from each other. Forexample, the two or more polynucleotide templates can include at leastone nucleic acid sequence that is substantially non-identical, orsubstantially non-complementary, to at least one other polynucleotidetemplate within the amplification reaction mixture.

In some embodiments, the amplifying optionally includes forming anamplification reaction mixture including a single continuous aqueousphase having two or more polynucleotide templates. Amplifying optionallyincludes forming two or more substantially monoclonal nucleic acidpopulations by clonally amplifying the two or more polynucleotidetemplates within the single aqueous phase. Optionally, the clonallyamplifying includes at least one round of RPA. Optionally, the clonallyamplifying includes at least one round of template walking.

In some embodiments, multiple different polynucleotide templates aredeposited or positioned to different sites prior to the amplifying. Forexample, amplified templates or extended first primers can be deposited,positioned or localized to different sites of an array.

In some embodiments, the amplifying results in the formation of at leasttwo substantially monoclonal nucleic acid populations (e.g., amplicons)in at least two different sites of the surface, which can then beanalyzed in situ using appropriate procedures.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems and kits) for preparing a surface.Optionally, the surface includes a plurality of sites, including a firstsite and a second site.

In some embodiments, the methods include forming a nucleic acid array onthe surface, wherein the forming includes linking a first nucleic acidto the first site and linking a second nucleic acid to the second site.The linking can optionally be performed using any of the methodsdisclosed herein, including for example by linking the nucleic acid to aprimer that is covalently attached to the surface.

In some embodiments, the methods include contacting at least the firstand second nucleic acids with a single reaction mixture includingreagents for nucleic acid synthesis. The reaction mixture can optionallyinclude any one or more of the components described herein. In someembodiments, the reaction mixture includes all of the componentsrequired to perform RPA. In some embodiments, the reaction mixtureincludes all of the components to perform template walking.

In some embodiments, the methods include forming a first amplicon at thefirst site and a second amplicon at the second site by replicating thefirst and second nucleic acids using the reagents for nucleic acidsynthesis in the reaction mixture. The replicating can include primerextension. The replicating can include one or more cycles of RPA. Thereplicating can include one or more cycles of template walking.

In some embodiments, the replicating includes at least one cycle of RPA.

In some embodiments, the replicating includes at least one cycle oftemplate walking.

In some embodiments, the replicating includes at least one cycle of RPAand at least one cycle of template walking.

In some embodiments, the replicating includes at least one round of RPA.

In some embodiments, the replicating includes at least one round oftemplate walking.

In some embodiments, the replicating includes at least one round of RPAand at least one round of template walking.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems and kits) for preparing a surface,comprising: (a) providing a surface with a plurality of sites, includinga first site and a second site; (b) forming a nucleic acid array on thesurface, wherein the forming includes linking a first nucleic acid tothe first site and linking a second nucleic acid to the second site;(c); and (d) forming a first amplicon at the first site and a secondamplicon at the second site by replicating the first and second nucleicacids using the reagents for nucleic acid synthesis in the reactionmixture.

In some embodiments, the disclosure relates generally to a method forpreparing a surface, comprising: providing a surface with a plurality ofsites, including a first site and a second site; forming a nucleic acidarray on the surface, wherein the forming includes linking a firstnucleic acid to the first site and linking a second nucleic acid to thesecond site; contacting at least the first and second nucleic acids witha single reaction mixture including reagents for nucleic acid synthesis;and forming a first substantially monoclonal amplicon at the first siteand a second substantially monoclonal amplicon at the second site byamplifying the first and second nucleic acids using the reagents fornucleic acid synthesis in the reaction mixture. Optionally, the firstand second sites remain in fluid communication during the amplifying.Optionally, the amplifying is performed without completely denaturingthe polynucleotides during the amplifying. For example, the disclosedmethods can include amplifying the at least two differentpolynucleotides via isothermal amplification. The amplifying can includeamplifying the at least two different polynucleotides undersubstantially isothermal conditions. Optionally, the amplifying isperformed without contacting the polynucleotides with chemicaldenaturants during the amplifying.

In some embodiments, at least one site of the plurality includes areaction well, channel, groove or chamber.

In some embodiments, at least one site of the plurality is linked to asensor.

In some embodiments, the sensor is capable of detecting a nucleotideincorporation occurring at or near the at least one site.

In some embodiments, the sensor includes a field effect transistor (FET)

In some embodiments, at least the first site, or the second site, orboth the first and second sites, include a primer linked to the surface.

In some embodiments, at least one of the sites of the plurality of sitescomprises a hydrophilic polymer matrix conformally disposed within awell operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix includes a hydrogel polymermatrix.

Optionally, the hydrophilic polymer matrix is a cured-in-place polymermatrix.

Optionally, the hydrophilic polymer matrix includes polyacrylamide,copolymers thereof, derivatives thereof, or combinations thereof.

Optionally, the polyacrylamide is conjugated with an oligonucleotideprimer.

Optionally, the well has a characteristic diameter in a range of 0.1micrometers to 2 micrometers.

Optionally, the well has a depth in a range of 0.01 micrometers to 10micrometers.

In some embodiments, the sensor includes a field effect transistor(FET). The FET can include an ion sensitive FET (ISFET), chemFET,bioFET, and the like.

In some embodiments, the FET is capable of detecting the presence of anucleotide incorporation byproduct at the at least one site.

In some embodiments, the FET is capable of detecting a chemical moietyselected from the group consisting of: hydrogen ions, pyrophosphate,hydroxyl ions, and the like.

In some embodiments, the methods (and related compositions, systems andkits) can include detecting the presence of one or more nucleotideincorporation byproducts at a site of the array, optionally using theFET.

In some embodiments, the methods can include detecting a pH changeoccurring at the site or within the at least one reaction chamber,optionally using the FET.

In some embodiments, the disclosed methods include introducing anucleotide into at least one of the sites in the plurality of sites; anddetecting an output signal from the sensor resulting from incorporationof the nucleotide into the sequencing primer. The output signal isoptionally based on a threshold voltage of the FET. In some embodiments,the FET includes a floating gate conductor coupled to the site.

In some embodiments, the FET includes a floating gate structurecomprising a plurality of conductors electrically coupled to one anotherand separated by dielectric layers, and the floating gate conductor isan uppermost conductor in the plurality of conductors.

In some embodiments, the floating gate conductor includes an uppersurface defining a bottom surface of the site.

In some embodiments, the floating gate conductor comprises anelectrically conductive material, and the upper surface of the floatinggate conductor includes an oxide of the electrically conductivematerial.

In some embodiments, the floating gate conductor is coupled to the atleast one reaction chamber via a sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture includes all of the componentsrequired to perform RPA.

In some embodiments, the reaction mixture includes all of the componentsrequired to perform template walking.

In some embodiments, the reaction mixture can include one or more solidor semi-solid supports. At least one of the supports can include one ormore instances of a first primer including a first primer sequence. Insome embodiments, at least one of the supports includes two or moredifferent primers attached thereto. For example, the at least onesupport can include at least one instance of the first primer and atleast one instance of a second primer.

Alternatively, in some embodiments, the reaction mixture does notinclude any supports. In some embodiments, the at least two differentpolynucleotide templates are amplified directly onto a surface of thesite or reaction chamber of the array.

In some embodiments, the reaction mixture can include a recombinase. Therecombinase can include any suitable agent that can promoterecombination between polynucleotide molecules. The recombinase can bean enzyme that catalyzes homologous recombination. For example, thereaction mixture can include a recombinase that includes, or is derivedfrom, a bacterial, eukaryotic or viral (e.g., phage) recombinase enzyme.

Optionally, the reaction mixture includes nucleotides that are notextrinsically labeled. For example, the nucleotides can be naturallyoccurring nucleotides, or synthetic analogs that do not includefluorescent moieties, dyes, or other extrinsic optically detectablelabels. Optionally, the reaction mixture includes nucleotides that arenaturally occurring nucleotides. Optionally, the nucleotides do notinclude groups that terminate nucleic acid synthesis (e.g., dideoxygroups, reversible terminators, and the like).

Optionally, the reaction mixture includes nucleotides that are naturallyoccurring nucleotides. Optionally, the nucleotides do not include groupsthat terminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like).

In some embodiments, the reaction mixture includes an enzyme that canbind a primer and a polynucleotide template to form a complex, or cancatalyze strand invasion of the polynucleotide template to form a D-loopstructure. In some embodiments, the reaction mixture includes one ormore proteins selected from the group consisting of: UvsX, RecA andRad51.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems and kits) for preparing a surface,comprising: (a) providing a surface with a plurality of sites, whereineach site is linked to a nucleic acid primer; (c) contacting the surfacewith a plurality of polynucleotide templates and binding at least one oftemplates to the surface; and amplifying at least one of the templatesonto the surface, thereby forming at least one substantially monoclonalpopulation of the amplified target polynucleotide sequence situated at asite on the surface.

In some embodiments, the disclosure relates generally to a method fornucleic acid amplification, comprising: (1) providing a surface having afirst site and a second site, the first site being operatively coupledto a first sensor and including a first template; the second site beingoperatively coupled to a second sensor and including a second template;(2) distributing to the first and the second sites a reaction mixture;and (3) forming a first amplicon by amplifying the first template at thefirst site; and forming a second amplicon by amplifying the secondtemplate at the second site.

In some embodiments, the amplifying includes at least one cycle of RPA.

In some embodiments, the amplifying includes at least one cycle oftemplate walking.

In some embodiments, the amplifying includes at least one cycle of RPAand at least one cycle of template walking.

In some embodiments, the amplifying includes at least one round of RPA.

In some embodiments, the amplifying includes at least one round oftemplate walking.

In some embodiments, the amplifying includes at least one round of RPAand at least one round of template walking.

In some embodiments, any some or all of the methods disclosed herein canresult in the production of a plurality of amplicons, at least some ofwhich amplicons include a clonally amplified nucleic acid population.The clonally amplified populations produced by the methods of thedisclosure can be useful for a variety of purposes. In some embodiments,the disclosed methods (and related compositions, systems and kits)optionally include further analysis and/or manipulation of the clonallyamplified populations (amplicons).

In some embodiments, any of the nucleic acid amplification methodsdisclosed herein can be conducted under conditions suitable to reducepolyclonality. In some embodiments, one source of polyclonality caninclude diffusion of a single-stranded or double stranded templatemolecule within the amplification reaction mixture from a first supportto a second support. The template molecule can include at least oneprimer binding site that can bind to a capture primer immobilized on thesecond support, and amplification at the second support can lead topolyclonality on the second support. In some embodiments, conditionssuitable to reduce polyclonality include altering or cleaving the primerbinding site on the diffusing template molecules, rendering the primerbinding site incompetent to hybridize to a capture primer immobilized ona support.

In some embodiments, the nucleic acid amplification reaction can beconducted using double-stranded nucleic acid template molecules havingat least one nucleotide that is susceptible to sequence-specific ornon-sequence-specific degradation by chemical reagents, enzymaticcleavage or photo-activated cleavage.

In some embodiments, one or both strands of the double-stranded nucleicacid template comprises at least one uracil base in the first or secondprimer binding site. Optionally, the uracil base can be degraded withuracil DNA glycosylase (UDG) thereby altering an intact primer bindingsite.

In some embodiments, any of the nucleic acid amplification methodsdisclosed herein can be conducted by hybridizing a primer binding siteof a soluble single-stranded template molecule with an RNAoligonucleotide (e.g., aptamer) having perfect or partialcomplementarity with the primer binding site to form a DNA/RNA duplex.Optionally, the DNA/RNA duplex can be contacted with a type IIP classrestriction enzyme that can cleave both the RNA and DNA strands in theduplex, thereby cleaving the primer binding site. Optionally, the typeIIP restriction enzyme include AvaII, AvrII, BanI, HaeIII, HinfI andTaq1 (Murray, et al., 2010 Nucleic Acids Research Vol. 38(22), pp.8257-8268, doi:10,1093/nar/gkq702). Alternatively, the DNA/RNA duplexcan be contacted with a duplex-specific nuclease (DSN). Optionally, theduplex-specific nuclease can cleave the DNA strand in a DNA/RNA duplex,thereby cleaving the primer binding site. Optionally, theduplex-specific nuclease can be isolated from, or derived from,hepatopancreas of Kamchatka crab (Shagin 2002 Genome Research12(12):1935-1942; Anisomova 2008 BMC Biochemistry 9:14).

In some embodiments, any of the nucleic acid amplification methodsdisclosed herein can be conducted by hybridizing a primer binding siteof a soluble single-stranded template molecule with a ribozyme (e.g.,ribonucleic acid enzyme). Optionally, SELEX (e.g., systematic evolutionof ligands by exponential enrichment) (Ellington and Szostak 1990 Nature346:818-822; Tuerk and Gold 1990 Science 249:505-510) can be employed toevolve and select a ribozyme that can bind a primer binding site andcleave single-stranded DNA. Optionally, the ribozyme comprises a groupII intron ribozyme from organelles of prokaryotes, protists, fungi,algae or plants (Griffith 1995 Chemistry and Biology, volume 2, issue11, pp. 761-770; Lehmann and Schmidt 2003 Critical Reviews inBiochemistry and Molecular Biology 38(3):249-303).

In some embodiments, any of the nucleic acid amplification methodsdisclosed herein can include at least one type of engineeredsequence-specific nuclease that can cleave spurious adaptor-dimer orprimer-dimer amplification products. In some embodiments, the engineeredsequence-specific nuclease comprises a sequence recognition module thatcan be engineered to bind specifically to a sequence within anadaptor-dimer or primer-dimer amplification product. In someembodiments, the engineered sequence-specific module can be joined to anuclease module that can cleave the adaptor-dimer or primer-dimeramplification product at a specific sequence or can catalyzenon-specific cleavage (for a review, see Gaj, et al., 2013 Trends inBiotechnology 31(7):397-405).

Optionally, the engineered sequence recognition module comprises aplurality of motifs from a TALE (transcription activator-like effector)protein. Optionally, the engineered TALE protein comprises a pluralityof DNA-binding domains from Xanthomonas. Optionally, the engineered TALEprotein can be joined to a nuclease, including an endonuclease (e.g.,FokI) (U.S. published application No. 2011/0145940; Boch 2009 Science326:1509-1512; Moscou 2009 Science 326:1501; Mak 2012 Science335:716-719; Deng 2012 Science 335:720-723).

Optionally, the engineered sequence recognition module comprises aplurality of sequence recognition motifs of a zinc-finger domain.Optionally, the plurality of engineered zinc-finger domains can bejoined to a nuclease, including an endonuclease (e.g., FokI) (U.S. Pat.Nos. 6,534,261, 7,013,219 and 7,220,719, all assigned to Sangamo, Inc.;Liu, et al., 1997 Proceeding of the National Academy of Science94:5525-5530).

Optionally, the engineered sequence recognition module comprises an RNAfrom a CRISPR/Cas system (Clustered Regularly Interspaced ShortPalindromic Repeats). Optionally, the engineered CRISPR/Cas RNA includesa dinucleotide-containing protospacer adjacent motif (PAM). Optionally,the CRISPR/Cas RNA can be complexed with a Cas protein having nucleaseactivity (Ishino, et al., 1987 Journal of Bacteriology169(12):5429-5433; Mojica, et al., 2000 Molecular Microbiology36(1):244-246; Jansen, et al., 2002 Molecular Microbiology43(6):1565-1575).

In some embodiments, amplicons produced according to the presentdisclosure can be subjected to downstream analysis methods such assequencing.

In some embodiments, the amplified nucleic acids can be further analyzed(e.g., sequencing) at the site of distribution without recovering andmoving the amplified products to a different site or surface foranalysis (e.g., sequencing).

In some embodiments, methods of downstream analysis include sequencingat least some of the plurality of amplicons in parallel. Optionally, themultiple templates/amplified templates/extended first primers situatedat different sites of the array are sequenced in parallel.

In some embodiments, the methods (and related compositions, systems, andkits) can further include sequencing an amplified template, orsequencing an extended primer, (e.g. an extended first primer, orextended second primer). The sequencing can include any suitable methodof sequencing known in the art. In some embodiments, the sequencingincludes sequencing by synthesis or sequencing by electronic detection(e.g., nanopore sequencing). In some embodiments, sequence includesextending a template or amplified template, or extending a sequencingprimer hybridized to a template or amplified template, via nucleotideincorporation by a polymerase. In some embodiments, sequencing includessequence a template or amplified template that is attached to a supportby contacting the template or extended primer with a sequencing primer,a polymerase, and at least one type of nucleotide. In some embodiments,the sequencing includes contacting the template, or amplified template,or extended primer, with a sequencing primer, a polymerase and with onlyone type of nucleotide that does not include an extrinsic label or achain terminating group.

For example, in some embodiments, amplifying is followed by sequencingthe amplified products in situ. The amplified product that is sequencedcan include an amplicon comprising a substantially monoclonal nucleicacid population. Optionally, monoclonal nucleic acid populations(amplicons) situated at different sites of the array are sequenced inparallel.

In some embodiments, the sequencing can include binding a sequencingprimer to the nucleic acids of at least two different amplicons, or atleast two different substantially monoclonal populations.

In some embodiments, the sequencing can include incorporating anucleotide into the sequencing primer using the polymerase. Optionally,the incorporating includes forming at least one nucleotide incorporationbyproduct.

Optionally, the nucleic acid to be sequenced is positioned at a site.The site can include a reaction chamber or well. The site can be part ofan array of similar or identical sites. The array can include atwo-dimensional array of sites on a surface (e.g., of a flowcell,electronic device, transistor chip, reaction chamber, channel, and thelike), or a three-dimensional array of sites within a matrix or othermedium (e.g., solid, semi-solid, liquid, fluid, and the like).

In some embodiments, the site is operatively coupled to a sensor. Themethod can include detecting the nucleotide incorporation using thesensor. Optionally, the site and the sensor are located in an array ofsites coupled to sensors.

In some embodiments, the site comprises a hydrophilic polymer matrixconformally disposed within a well operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix includes a hydrogel polymermatrix.

Optionally, the hydrophilic polymer matrix is a cured-in-place polymermatrix.

Optionally, the hydrophilic polymer matrix includes polyacrylamide,copolymers thereof, derivatives thereof, or combinations thereof.

Optionally, the polyacrylamide is conjugated with an oligonucleotideprimer.

Optionally, the well has a characteristic diameter in a range of 0.1micrometers to 2 micrometers.

Optionally, the well has a depth in a range of 0.01 micrometers to 10micrometers.

In some embodiments, the sensor includes a field effect transistor(FET). The FET can include an ion sensitive FET (ISFET).

In some embodiments, the methods (and related compositions, systems andkits) can include detecting the presence of one or more nucleotideincorporation byproducts at a site of the array, optionally using theFET.

In some embodiments, the methods can include detecting a pH changeoccurring at the site or within the at least one reaction chamber,optionally using the FET.

In some embodiments, the disclosed methods include introducing anucleotide into the site; and detecting an output signal from the sensorresulting from incorporation of the nucleotide into the sequencingprimer. The output signal is optionally based on a threshold voltage ofthe FET. In some embodiments, the FET includes a floating gate conductorcoupled to the site.

In some embodiments, the FET includes a floating gate structurecomprising a plurality of conductors electrically coupled to one anotherand separated by dielectric layers, and the floating gate conductor isan uppermost conductor in the plurality of conductors.

In some embodiments, the floating gate conductor includes an uppersurface defining a bottom surface of the site.

In some embodiments, the floating gate conductor comprises anelectrically conductive material, and the upper surface of the floatinggate conductor includes an oxide of the electrically conductivematerial.

In some embodiments, the floating gate conductor is coupled to the atleast one reaction chamber via a sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture includes all of the componentsrequired to perform RPA.

In some embodiments, the reaction mixture includes all of the componentsrequired to perform template walking.

In some embodiments, the reaction mixture can include one or more solidor semi-solid supports. At least one of the supports can include one ormore instances of a first primer including a first primer sequence. Insome embodiments, at least one of the supports includes two or moredifferent primers attached thereto. For example, the at least onesupport can include at least one instance of the first primer and atleast one instance of a second primer.

Alternatively, in some embodiments, the reaction mixture does notinclude any supports. In some embodiments, the at least two differentpolynucleotide templates are amplified directly onto a surface of thesite or reaction chamber of the array.

In some embodiments, the reaction mixture can include a recombinase. Therecombinase can include any suitable agent that can promoterecombination between polynucleotide molecules. The recombinase can bean enzyme that catalyzes homologous recombination. For example, thereaction mixture can include a recombinase that includes, or is derivedfrom, a bacterial, eukaryotic or viral (e.g., phage) recombinase enzyme.

Optionally, the reaction mixture includes nucleotides that are notextrinsically labeled. For example, the nucleotides can be naturallyoccurring nucleotides, or synthetic analogs that do not includefluorescent moieties, dyes, or other extrinsic optically detectablelabels. Optionally, the reaction mixture includes nucleotides that arenaturally occurring nucleotides. Optionally, the nucleotides do notinclude groups that terminate nucleic acid synthesis (e.g., dideoxygroups, reversible terminators, and the like).

Optionally, the reaction mixture includes nucleotides that are naturallyoccurring nucleotides. Optionally, the nucleotides do not include groupsthat terminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like).

In some embodiments, the reaction mixture includes an enzyme that canbind a primer and a polynucleotide template to form a complex, or cancatalyze strand invasion of the polynucleotide template to form a D-loopstructure. In some embodiments, the reaction mixture includes one ormore proteins selected from the group consisting of: UvsX, RecA andRad51.

In some embodiments, the reaction mixture can include a recombinaseaccessory protein, for example UvsY.

In some embodiments, the reaction mixture can include a single strandedbinding protein (SSBP).

In some embodiments, the reaction mixture can include a polymerase. Thepolymerase optionally has, or lacks, exonuclease activity. In someembodiments, the polymerase has 5′ to 3′ exonuclease activity, 3′ to 5′exonuclease activity, or both. Optionally, the polymerase lacks any oneor more of such exonuclease activities.

In some embodiments, the polymerase has strand displacing activity.

In some embodiments, the reaction mixture can include a diffusionlimiting agent. The diffusion limiting agent can be any agent that iseffective in preventing or slowing the diffusion of one or more of thepolynucleotide templates and/or one or more of the amplificationreaction products through the reaction mixture.

In some embodiments, the reaction mixture can include a sieving agent.The sieving agent can be any agent that is effective in sieving one ormore polynucleotides present in the reaction mixture, such as forexample amplification reaction products and/or polynucleotide templates.In some embodiments, the sieving agent restricts or slows the migrationof polynucleotide amplification productions through the reactionmixture.

In some embodiments, the reaction mixture can include a crowding agent.

In some embodiments, the reaction mixture includes both a crowding agentand a sieving agent.

In some embodiments, the disclosed methods include contacting each ofthe at least two polynucleotides with a recombinase, a support attachedto a plurality of first oligonucleotide primers, the firstoligonucleotide primers being at least partially complementary to atleast some portion of the polynucleotides, a polymerase, and a pluralityof nucleotides, in any order and in any combination.

In some embodiments, the at least two different polynucleotides includea forward strand containing a first primer binding site, and theamplifying within the at least two sites (or within the at least tworeaction chambers) optionally includes binding a first primer to a firstprimer binding site to form a first primer-template duplex within thesites or reaction chambers. Optionally, the binding of the first primerto the at least two different polynucleotide templates is mediated by arecombinase. For example, the amplifying can include forming anucleoprotein complex containing a recombinase and first primer.Optionally, the first primer is attached to a surface of the site orreaction chamber. In some embodiments, the amplifying within the sitesor reaction chambers includes: forming a first nucleoprotein complex (or“first nucleoprotein filament”). The amplifying optionally furtherincludes contacting at least one of the polynucleotides in the sites orreaction chambers with the first nucleoprotein filament, a polymeraseand a plurality of nucleotides, in any order or combination.

Optionally, discrete supports (e.g., beads), each containing a pluralityof first primers, are individually distributed into the reactionchambers or sites prior to the amplifying, and the amplifying includesamplifying one of the at least two different polynucleotides onto thesupport within the site or reaction chamber. In some embodiments, anyone of the distributing and/or contacting steps can be repeated prior tothe amplifying, optionally to increase yield and/or number of sites orreaction chambers yielding monoclonal product.

Optionally, the amplifying includes extending the first primer of thefirst primer-template duplex using a polymerase within the reactionchamber, thereby forming an extended first primer. Optionally, extendingthe first primer displaces the reverse strand from the forward strand.The extended first primer optionally includes a second primer bindingsite.

Optionally, the amplifying includes a step of reverse synthesiscomprising binding a second primer to the second primer binding site ofthe extended first primer, and extending the second primer to form asecond primer-template duplex. Optionally, the binding of the secondprimer to the polynucleotide templates is mediated by a recombinase. Forexample, the amplifying can include forming a nucleoprotein complexcontaining a recombinase and a second primer. Optionally, the secondprimer is attached to a surface of the site or reaction chamber. In someembodiments, the amplifying within the sites or reaction chambersincludes: forming a second nucleoprotein complex (or “secondnucleoprotein filament”). The amplifying optionally further includescontacting at least one of the polynucleotide templates, or at least oneof the extended first primers in the sites or reaction chambers with thesecond nucleoprotein filament, a polymerase and a plurality ofnucleotides, in any order or combination.

Optionally, the amplifying includes extending the first primer-templateduplex, the second primer-template duplex, or both, using a polymerase.The polymerase can have strand-displacing activity.

In some embodiments, the methods (and related compositions, systems andkits) can include depositing, positioning or localizing at least onesubstantially monoclonal population at a site. The site can form part ofan array of sites.

Optionally, at least one of the sites includes a reaction chamber,support, particle, microparticle, sphere, bead, filter, flowcell, well,groove, channel reservoir, gel or inner wall of a tube.

In some embodiments, at least one site comprises a hydrophilic polymermatrix conformally disposed within a well operatively coupled to thesensor.

Optionally, the hydrophilic polymer matrix includes a hydrogel polymermatrix.

Optionally, the hydrophilic polymer matrix is a cured-in-place polymermatrix.

Optionally, the hydrophilic polymer matrix includes polyacrylamide,copolymers thereof, derivatives thereof, or combinations thereof.

Optionally, the polyacrylamide is conjugated with an oligonucleotideprimer.

Optionally, the well has a characteristic diameter in a range of 0.1micrometers to 2 micrometers.

Optionally, the well has a depth in a range of 0.01 micrometers to 10micrometers.

In some embodiments, the sensor includes a field effect transistor(FET). The FET can include an ion sensitive FET (ISFET).

In some embodiments, the methods (and related compositions, systems andkits) can include detecting the presence of one or more nucleotideincorporation byproducts at a site of the array, optionally using theFET.

In some embodiments, the methods can include detecting a pH changeoccurring within the at least one reaction chamber, optionally using theFET.

In some embodiments, the disclosed methods include introducing anucleotide into the site; and detecting an output signal from the sensorresulting from incorporation of the nucleotide into the sequencingprimer. The output signal is optionally based on a threshold voltage ofthe FET. In some embodiments, the FET includes a floating gate conductorcoupled to the site.

In some embodiments, the FET includes a floating gate structurecomprising a plurality of conductors electrically coupled to one anotherand separated by dielectric layers, and the floating gate conductor isan uppermost conductor in the plurality of conductors.

In some embodiments, the floating gate conductor includes an uppersurface defining a bottom surface of the site.

In some embodiments, the floating gate conductor comprises anelectrically conductive material, and the upper surface of the floatinggate conductor includes an oxide of the electrically conductivematerial.

In some embodiments, the floating gate conductor is coupled to the atleast one reaction chamber via a sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

Optionally, the plurality of different polynucleotide templates (oramplified polynucleotides) includes at least one nucleic acid containinga selectively cleavable moiety.

Optionally, the selectively cleavable moiety includes uracil.

Optionally, methods for nucleic acid amplification further includecleaving the cleavable moiety with a cleaving agent.

Optionally, the cleaving can be performed prior to the amplifying, forexample before forming the reaction mixture.

Optionally, the cleaving can be performed after the amplifying, e.g.,after the nucleic acid template(s) are amplified.

Optionally, the reaction mixture includes at least one primer containinga cleavable moiety.

Optionally, methods for nucleic acid amplification further includecleaving the cleavable moiety with a cleaving agent.

Optionally, the plurality of different polynucleotides includes aplurality of amplicons.

Optionally, the plurality of different polynucleotides includes aplurality of different amplicons.

In some embodiments, amplification can be practiced using any of themethods, compositions, systems and kits disclosed in U.S. ProvisionalApplication No. 61/792,247, filed Mar. 15, 2013, incorporated byreference herein in its entirety.

In some embodiments, amplicons produced according to the presentdisclosure can be subjected to downstream analysis methods such asquantification. For example, in some embodiments, the numbers ofamplicons exhibiting certain desired characteristics can be detected andoptionally quantified.

In some embodiments, in the disclosed methods the amplified nucleicacids can optionally be subjected to additional steps of downstreamanalysis.

In some embodiments involving amplification of different polynucleotidetemplates onto discrete and separate supports, the methods can includedetermining which of the discrete supports (e.g., beads), includeamplicons. Similarly, in embodiments where the templates are distributedinto an array prior to the amplifying, the methods can includedetermining which sites of the array include amplicons, and canoptionally further include counting the number of sites that includeamplicons. The presence of amplicons at supports or sites can optionallybe detected determined using DNA-based detection procedures such as UVabsorbance, staining with DNA-specific dyes, TAQMAN® assays, qPCR,hybridization to fluorescent probes, and the like. In some embodiments,the methods can include determining which bead supports (or sites of anarray) have received substantially monoclonal amplicons. For example,the bead supports (or array sites) can be analyzed to determine whichsupports or sites can produce a detectable and coherent (i.e.,analyzable) sequence-dependent signal.

In some embodiments, the disclosed methods include additional steps ofdownstream analysis that provide the same type of information previouslyobtained through conventional techniques such as digital PCR or digitalRPA as described, for example, in Shen 2011 Analytical Chemistry83:3533-3540; U.S. published applications 2012/0264132 and 2012/0329038;all of which are incorporated by reference in their entireties. DigitalPCR (dPCR) is a refinement of conventional polymerase chain reaction(PCR) methods which can be used to directly quantify and clonallyamplify nucleic acids (including DNA, cDNA, methylated DNA, or RNA). Onedifference between dPCR and traditional PCR lays in the method ofmeasuring nucleic acids amounts. Both PCR and dPCR carry out onereaction per single sample, dPCR also carries out a single reactionwithin a sample, however the sample is separated into a large number ofpartitions and the reaction is carried out in each partitionindividually. This separation allows for sensitive measurement ofnucleic acid amounts. dPCR has been demonstrated as useful for studyingvariations in gene sequences, such as copy number variation or pointmutations.

In contract to the instant methods, dPCR typically requires partitioningof the sample prior to amplification; in contrast, several of theembodiments disclosed herein provide for parallel amplification ofdifferent templates within a single continuous phase of a reactionmixture without need for partitioning. In dPCR, a sample is typicallypartitioned so that individual nucleic acid molecules within the sampleare localized and concentrated within many separate regions. The sampleis fractionated by the simple process of dilution so that each fractioncontains approximately one copy of DNA template or less. By isolatingindividual DNA templates this process effectively enriches DNA moleculesthat were present at very low levels in the original sample. Thepartitioning of the sample facilitates counting of molecules usingPoisson statistics. As a result, each partition will contain “0” or “1”molecule(s), or a negative or positive reaction, respectively. While thestarting copy number of a molecule is proportional to the number ofamplification cycles in conventional PCR, dPCR is not typicallydependent on the number of amplification cycles to determine the initialsample amount.

Conventional methods of dPCR analysis typically utilize fluorescentprobes and light based detection methods to identify the products ofamplification. Such approaches require sufficient amplification of thetarget molecules to generate enough signal to be detectable but can leadto additional error or bias.

In those embodiments of the present disclosure involving distribution ofnucleic acid templates into the wells of an isFET array and subsequentamplification of templates inside the wells of the array, an optionalstep of downstream analysis can be performed after the amplificationthat quantifies the number of sites or wells that include amplificationproduct. In some embodiments, the products of the nucleic acidamplification reactions can be detected in order to count the number ofsites or wells that include an amplified template.

For example, in some embodiments the disclosure relates generally tomethods of nucleic acid analysis, comprising: providing a sampleincluding a first number of polynucleotides; and distributing singlepolynucleotides of the sample into different sites in an array of sites.

Optionally, the methods can further include forming substantiallymonoclonal nucleic acid populations by amplifying the singlepolynucleotides within their respective sites.

Optionally, the sites remain in fluid communication during theamplifying.

Optionally, the amplifying includes partially denaturing the template.

Optionally, the amplifying includes subjecting the template to partiallydenaturing temperatures. In some embodiments, the template includes alow-melt sequence including a primer binding site, which is renderedsingle stranded when the template is subjected to partially denaturingtemperatures.

Optionally, the amplifying includes partially denaturing the template.

Optionally, the amplifying includes contacting at least two differenttemplates at two different sites of the array with a single reactionmixture for nucleic acid amplification.

Optionally, the reaction mixture includes a recombinase.

Optionally, the reaction mixture includes at least one primer includinga “drag-tag”.

Optionally, the amplifying includes performing at least oneamplification cycle that includes partially denaturing the template,hybridizing a primer to the template, and extending the primer in atemplate-dependent fashion. Optionally, the amplifying includesisothermally amplifying. In some embodiments, the amplifying isperformed under substantially isothermal conditions.

In some embodiments, the percentage of sites containing one or moretemplate molecules is greater than 50% and less than 100%.

In some embodiments, the disclosed methods can further include detectinga change in ion concentration in at least one of the sites as a resultof the at least one amplification cycle.

In some embodiments, the disclosed methods can further includequantitating an initial amount of target nucleic acid.

Some examples of array-based digital PCR using ion-based sensingtechnology can be found, for example, in U.S. Provisional Appl. No.61/635,584, filed Apr. 19, 2012, hereby incorporated by reference in itsentirety.

In some embodiments, the disclosure relates generally to methods fordetection of a target nucleic acid comprising: fractionating a sampleinto a plurality of sample volumes wherein more than 50% of thefractions contain no more than 1 target nucleic acid molecule per samplevolumes; subjecting the plurality of sample volumes to conditions foramplification, wherein the conditions include partially denaturingconditions; detecting a change in ion concentration in a sample volumewherein a target nucleic acid is present; counting the number offractions with an amplified target nucleic acid; and determining thequantity of target nucleic acid in the sample. The change in ionconcentration may be an increase in ion concentration or may be adecrease in ion concentration. In some embodiments, the method mayfurther include combining a sample with bead. In some embodiments, themethod may include loading the sample on a substrate wherein thesubstrate includes at least one well.

In some embodiments, subjecting the target nucleic acids to partiallydenaturing conditions includes contacting the target nucleic acidmolecules in their respective sample volumes with a recombinase and apolymerase under RPA conditions.

In some embodiments, subjecting the target nucleic acids to partiallydenaturing conditions includes subjecting the target nucleic acidmolecules to partially denaturing temperatures.

In some embodiments, the disclosure relates generally to a method forperforming absolute quantification of a nucleic acid comprising:diluting a sample containing an initial number of nucleic acid templatesand distributing the nucleic acid templates of the sample into aplurality of sites of an array, wherein the percentage of sitescontaining one or more nucleic acid templates is greater than 50% andless than 100%; subjecting the plurality of sites to at least oneamplification cycle, wherein the amplification cycle is performedaccording to any of the amplification methods disclosed herein;detecting a change in ion concentration in at least one of the pluralityof sample volumes as a result of the at least one amplification cycle;and quantitating an initial amount of nucleic acid templates. The changein ion concentration may be an increase in ion concentration, a decreasein ion concentration, a change in pH, may involve the detection of apositive ion such as a hydrogen ion, a negative ion such as apyrophosphate molecule, or both positive and negative ion.

In some embodiments, the disclosure also relates generally to methods(and related compositions, systems and kits) for linking individualmembers of a population of nucleic acid templates to different supportsin a plurality of supports, or to different sites in a plurality ofsites using recombinase-mediated strand exchange. These methods,compositions, systems and kits can be useful for generating populationsof immobilized amplicons amenable to manipulation in applicationsrequiring different amplicons to be individually accessible ordistinguishable. In some embodiments, the plurality of discretesupports, or the plurality of sites in the array, each include a captureprimer. Immobilization of individual templates to individual supports(or to individual sites of the array) can be achieved by contacting thetemplates with the supports or sites in the presence of a primer(“fusion primer”). In some embodiments, the fusion primer includes atarget-specific portion that is complementary to a portion of thetemplate, and a universal primer-binding site that is complementary toat least some portion of the capture primer of the support or site.Optionally, the contacting is performed in the presence of RPAcomponents. The RPA components can include a recombinase. The RPAcomponents can include a strand-displacing polymerase. In someembodiments, the fusion primer is recombined into the template viarecombinase-mediated strand exchange, thereby forming a template:primeradduct including a universal primer-binding site. In some embodiments,the capture primer is then recombined into the universal primer-bindingsite, forming an immobilized template that is attached to the support orsite.

In some embodiments of bead-based amplification, a library of fusionprimers, each including a different target-specific portion and a commonuniversal primer-binding site, is contacted with a plurality oftemplates and a plurality of supports in a reaction mixture including apolymerase and a strand-displacing polymerase. The template library isthen attached to the plurality of supports by subjecting the mixture toRPA conditions, thereby generating a plurality of supports each attachedto a different template.

In some embodiments of array-based amplification, a library of fusionprimers, each including a different target-specific portion and a commonuniversal primer-binding site, is contacted with a plurality oftemplates and a surface including a plurality of sites in a reactionmixture including a polymerase and a strand-displacing polymerase. Atleast some of the plurality of sites include a universal capture primer.The template library is then attached to the plurality of sites of thesurface by subjecting the mixture to RPA conditions, thereby generatinga plurality of supports each attached to a different template.

In some embodiments, the disclosure relates generally to compositions(and relate methods for making and using said compositions) comprisingreagents for amplifying one or more nucleic acid templates in parallelusing partially denaturing conditions.

In some embodiments, the compositions can include any of the componentsdescribed herein for performing RPA.

In some embodiments, the compositions can include any of the componentsdescribed herein for performing template walking.

In some embodiments, the disclosure relates generally to compositionsand systems for nucleic acid amplification, comprising: a surfaceincluding a first site and a second site; and a nucleic acidamplification reaction mixture, wherein the mixture is in contact withthe first and second sites.

In some embodiments, the reaction mixture includes a recombinase.

In some embodiments, the first site is operatively coupled to a firstsensor, and the second site being operatively linked to a second sensor.

In some embodiments, the first and second sites are operatively linkedto the same sensor.

Optionally, the first site includes a first substantially monoclonalpopulation of nucleic acid. The second site optionally includes a secondsubstantially monoclonal population of nucleic acids

In some embodiments, the disclosed compositions comprise: a surfaceincluding a first site and a second site, wherein the first siteincludes a first substantially monoclonal population of nucleic acidsand the second site includes a second substantially monoclonalpopulation of nucleic acids; and a nucleic acid amplification reactionmixture, wherein the mixture is in contact with the first and secondsites.

In some embodiments, the compositions include an array of sites,including a first site containing (e.g., linked to) a first captureprimer, and a second site containing (e.g., linked to) a second captureprimer.

In some embodiments, at least one site of the plurality includes areaction well, channel, groove or chamber.

In some embodiments, at least one site of the plurality is linked to asensor.

In some embodiments, the sensor is capable of detecting a nucleotideincorporation occurring at or near the at least one site.

In some embodiments, the sensor includes a field effect transistor (FET)

In some embodiments, at least the first site, or the second site, orboth the first and second sites, include a capture primer linked to thesurface.

In some embodiments, at least one of the sites of the plurality of sitescomprises a hydrophilic polymer matrix conformally disposed within awell operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix includes a hydrogel polymermatrix.

Optionally, the hydrophilic polymer matrix is a cured-in-place polymermatrix.

Optionally, the hydrophilic polymer matrix includes polyacrylamide,copolymers thereof, derivatives thereof, or combinations thereof.

Optionally, the polyacrylamide is conjugated with an oligonucleotideprimer.

Optionally, the well has a characteristic diameter in a range of 0.1micrometers to 2 micrometers.

Optionally, the well has a depth in a range of 0.01 micrometers to 10micrometers.

In some embodiments, the sensor includes a field effect transistor(FET). The FET can include an ion sensitive FET (ISFET), chemFET,bioFET, and the like.

In some embodiments, the FET is capable of detecting the presence of anucleotide incorporation byproduct at the at least one site.

In some embodiments, the FET is capable of detecting a chemical moietyselected from the group consisting of: hydrogen ions, pyrophosphate,hydroxyl ions, and the like.

In some embodiments, the methods (and related compositions, systems andkits) can include detecting the presence of one or more nucleotideincorporation byproducts at a site of the array, optionally using theFET.

In some embodiments, the methods can include detecting a pH changeoccurring at the site or within the at least one reaction chamber,optionally using the FET.

In some embodiments, the disclosed methods include introducing anucleotide into at least one of the sites in the plurality of sites; anddetecting an output signal from the sensor resulting from incorporationof the nucleotide into the sequencing primer. The output signal isoptionally based on a threshold voltage of the FET. In some embodiments,the FET includes a floating gate conductor coupled to the site.

In some embodiments, the FET includes a floating gate structurecomprising a plurality of conductors electrically coupled to one anotherand separated by dielectric layers, and the floating gate conductor isan uppermost conductor in the plurality of conductors.

In some embodiments, the floating gate conductor includes an uppersurface defining a bottom surface of the site.

In some embodiments, the floating gate conductor comprises anelectrically conductive material, and the upper surface of the floatinggate conductor includes an oxide of the electrically conductivematerial.

In some embodiments, the floating gate conductor is coupled to the atleast one reaction chamber via a sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture includes all of the componentsrequired to perform RPA.

In some embodiments, the reaction mixture includes all of the componentsrequired to perform template walking.

In some embodiments, the reaction mixture can include one or more solidor semi-solid supports. At least one of the supports can include one ormore instances of a first primer including a first primer sequence. Insome embodiments, at least one of the supports includes two or moredifferent primers attached thereto. For example, the at least onesupport can include at least one instance of the first primer and atleast one instance of a second primer.

In some embodiments, a plurality of first supports includes two or moresub-population of first supports. Different sub-populations of firstsupports can optionally be attached with variations of the first primersequence, or alternatively the supports of each sub-population can beattached with different primers. For example, a first sub-population offirst supports can be attached with a first primer sequence (e.g., firstprimer sequence “A”), and a second sub-population of first supports canbe attached with a variation of the first primer sequence (e.g., firstprimer sequence “B”) that differs from the first primer sequence “A” atone or more positions. Optionally, other sub-populations of firstsupports can have first primer sequences with other variations, forexample first primer sequence “C”, “D” or others. Optionally, aplurality of double stranded nucleic acid templates comprises two ormore sub-populations of double stranded nucleic acid templates havingvariations of the first primer binding site that binds with the firstprimer sequence “A” or “B” attached to the sub-populations of firstsupports.

Alternatively, in some embodiments, the reaction mixture does notinclude any supports. In some embodiments, the at least two differentpolynucleotide templates are amplified directly onto a surface of thesite or reaction chamber of the array.

In some embodiments, the reaction mixture can include a recombinase. Therecombinase can include any suitable agent that can promoterecombination between polynucleotide molecules. The recombinase can bean enzyme that catalyzes homologous recombination. For example, thereaction mixture can include a recombinase that includes, or is derivedfrom, a bacterial, eukaryotic or viral (e.g., phage) recombinase enzyme.

Optionally, the reaction mixture includes nucleotides that are notextrinsically labeled. For example, the nucleotides can be naturallyoccurring nucleotides, or synthetic analogs that do not includefluorescent moieties, dyes, or other extrinsic optically detectablelabels. Optionally, the reaction mixture includes nucleotides that arenaturally occurring nucleotides. Optionally, the nucleotides do notinclude groups that terminate nucleic acid synthesis (e.g., dideoxygroups, reversible terminators, and the like).

Optionally, the reaction mixture includes nucleotides that are naturallyoccurring nucleotides. Optionally, the nucleotides do not include groupsthat terminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like).

In some embodiments, the reaction mixture includes an enzyme that canbind a primer and a polynucleotide template to form a complex, or cancatalyze strand invasion of the polynucleotide template to form a D-loopstructure. In some embodiments, the reaction mixture includes one ormore proteins selected from the group consisting of: UvsX, RecA andRad51.

In some embodiments, methods of amplifying can include performing“template walking” as described in U.S. Patent Publ. No. 2012/0156728,published Jun. 21, 2012, incorporated by reference herein in itsentirety. For example, in some embodiments, the disclosure relatesgenerally to methods, compositions, systems, apparatuses and kits forclonally amplifying one or more nucleic acid templates to form clonallyamplified populations of nucleic acid templates. Any amplificationmethod described herein optionally comprises repeated cycles of nucleicacid amplification. A cycle of amplification optionally comprises (a)hybridization of primer to a template strand, (b) primer extension toform a first extended strand, (c) partial or incomplete denaturation ofthe extended strand from the template strand. The primer that hybridizesto the template strand (designated “forward” primer for convenience) isoptionally immobilized on or to a support. The support is for examplesolid or semi-solid. Optionally, the denatured portion of the templatestrand from step (c) is free to hybridize with a different forwardprimer in the next amplification cycle. In an embodiment, primerextension in a subsequent amplification cycle involves displacement ofthe first extended strand from the template strand. A second “reverse”primer can for example be included which hybridizes to the 3′ end of thefirst extended strand. The reverse primer is optionally not immobilized.

In an embodiment, the templates are amplified using primers immobilizedon/to one or more solid or semi-solid supports. Optionally the supportcomprises immobilized primers that are complementary to a first portionof a template strand. Optionally, the support does not significantlycomprise an immobilized primer that is homologous to a secondnon-overlapping portion of the same template strand. The two portionsare non-overlapping if they do not contain any subportions thathybridize to each other or to a complement thereof. In another example,the support optionally does not significantly comprise an immobilizedprimer that can hybridize to the complement of the template strand).

Optionally, a plurality of nucleic acid templates are amplifiedsimultaneously in a single continuous liquid phase in the presence ofone or more supports, where each support comprises one or moreimmobilization sites. In an embodiment, each template is amplified togenerate a clonal population of amplicons, where individual clonalpopulations are immobilized within or on a different support orimmobilization site from other amplified populations. Optionally, theamplified populations remain substantially clonal after amplification.

A template is for example amplified to generate clonal populations whichcomprise template-homologous strands (called “template strands” or“reverse strands” herein) and/or template-complementary strands (called“primer strands” or “forward strands” herein). In an embodimentclonality is maintained in the resulting amplified nucleic acidpopulations by maintaining association between template strands and itsprimer strands, thereby effectively associating or “tethering”associated clonal progeny together and reducing the probability ofcross-contamination between different clonal populations. Optionally,one or more amplified nucleic acids in the clonal population is attachedto a support. A clonal population of substantially identical nucleicacids can optionally have a spatially localized or discrete macroscopicappearance. In an embodiment a clonal population can resemble a distinctspot or colony (e.g., when distributed in a support, optionally on theouter surface of the support).

In some embodiments, the disclosure relates generally to novel methodsof generating a localized clonal population of clonal amplicons,optionally immobilized in/to/on one or more supports. The support canfor example be solid or semisolid (such as a gel or hydrogel). Theamplified clonal population is optionally attached to the support'sexternal surface or can also be within the internal surfaces of asupport (e.g., where the support has a porous or matrix structure).

In some embodiments, amplification is achieved by multiple cycles ofprimer extension along a template strand of interest (also called a“reverse” strand). For convenience, a primer that hybridizes to thetemplate strand of interest is termed a “forward” primer, and isoptionally extended in template-dependent fashion to form a “forward”strand that is complementary to the template strand of interest. In somemethods, the forward strand is itself hybridized by a second primertermed the “reverse” primer, which is extended to form a new templatestrand (also called a reverse strand). Optionally, at least a portion ofthe new template strand is homologous to the original template(“reverse”) strand of interest.

As mentioned, one or more primers can be immobilized in/on/to one ormore supports. Optionally, one primer is immobilized by attachment to asupport. A second primer can be present and is optionally notimmobilized or attached to a support. Different templates can forexample be amplified onto different supports or immobilization sitessimultaneously in a single continuous liquid phase to form clonalnucleic acid populations. A liquid phase can be considered continuous ifany portion of the liquid phase is in fluid contact or communicationwith any other portion of the liquid body. In another example, a liquidphase can be considered continuous if no portion is entirely subdividedor compartmentalized or otherwise entirely physically separated from therest of the liquid body. Optionally, the liquid phase is flowable.Optionally, the continuous liquid phase is not within a gel or matrix.In other embodiments, the continuous liquid phase is within a gel ormatrix. For example the continuous liquid phase occupies pores, spacesor other interstices of a solid or semisolid support.

Where the liquid phase is within a gel or matrix, one or more primersare optionally immobilized on a support. Optionally the support is thegel or matrix itself. Alternatively the support is not the gel or matrixitself. In an example one primer is immobilized on a solid supportcontained within a gel and is not immobilized to gel molecules. Thesupport is for example in the form of a planar surface or one or moremicroparticles. Optionally the planar surface or plurality ofmicroparticles comprises forward primers having substantially identicalsequence. In an embodiment, the support does not contain significantamounts of a second different primer. Optionally, a secondnon-immobilized primer is in solution within the gel. The secondnon-immobilized primer for example binds to a template strand (i.e.,reverse strand), whereas the immobilized primer binds to a forwardstrand.

An embodiment of template walking includes a method of primer extension,comprising: (a) a primer-hybridization step, (b) an extension step, and(c) a walking step. Optionally, the primer-hybridization step compriseshybridizing a first primer molecule (“first forward primer”) to acomplementary forward-primer-binding sequence (“forward PBS”) on anucleic acid strand (“reverse strand”). Optionally the extension stepcomprises generating an extended first forward strand that is afull-length complement of the reverse strand and is hybridized thereto.The extended first forward strand is for example generated by extendingthe first forward primer molecule in template-dependent fashion usingthe reverse strand as template. Optionally the walking step compriseshybridizing a second primer (“second forward primer”) to the forward PBSwhere the reverse strand is also hybridized to the first forward strand.For example, the walking step comprises denaturing at least a portion ofthe forward PBS from the forward strand (“free portion”), where anotherportion of the reverse strand remains hybridized to the forward strand.

In an embodiment, the primer extension method is an amplification methodthat includes template walking, in which any one or more steps ofprimer-hybridization, extension and/or walking are repeated at leastonce. For example, the method can comprise amplifying the forward strandby one or more amplification cycles. An amplification cycle optionallycomprises extension and walking. An exemplary amplification cyclecomprises or consists essentially of extension followed by walking.Optionally, the second forward primer of a first amplification cycleacts as the first forward primer of a subsequent amplification cycle.For example, the second forward primer of a walking step in a firstamplification cycle acts as the first forward primer of an extensionstep of a subsequent amplification cycle.

Optionally, the method of primer extension or amplification furthercomprises extending or amplifying the reverse strand by (a) hybridizinga first reverse primer molecule to a complementaryreverse-primer-binding sequence (“reverse PBS”) on an extended forwardstrand; (b) generating an extended first reverse strand that is afull-length complement of the forward strand and hybridized thereto, byextending the first reverse primer molecule in template-dependentfashion using the forward strand as template; and (c) hybridizing asecond primer (“second reverse primer”) to the reverse PBS where theforward strand is also hybridized to the first reverse strand. One ormore repetitions of steps (b)-(c) are optionally performed, wherein thesecond reverse primer of step (c) is the first reverse primer ofrepeated step (b); and wherein a substantial proportion of forwardstrands are hybridized to reverse strands at all times during or betweensaid one or more repetitions. In embodiments, the substantial proportionis optionally at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or 99%.

Optionally, during amplification the reverse strand and/or forwardstrand is not exposed to totally-denaturing conditions that would resultin complete separation of a significant fraction (e.g., more than 10%,20%, 30%, 40% or 50%) of a large plurality of strands from theirextended and/or full-length complements.

In an embodiment a substantial proportion of forward and/or reversestrands are optionally hybridized to extended and/or full-lengthcomplements at all times during or between one or more amplificationcycles (e.g., 1, 5, 10, 20, or all amplification cycles performed). Inembodiments, the substantial proportion of strands is optionally atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of strands. In anembodiment this is achieved by maintaining the amplification reaction ata temperature higher than the T_(m) of unextended primers, but lowerthan the T_(m) of the primer-complementary strands. For example,amplification conditions are kept within a temperature that is higherthan the T_(m) of unextended forward primers, but lower than the T_(m)of extended or full-length reverse strands. Also for example,amplification conditions are kept within a temperature that is higherthan the T_(m) of unextended reverse primers, but lower than the T_(m)of extended or full-length forward strands.

Optionally, one or more forward primers, and/or one or more reverseprimers are breathable, e.g., have a low T_(m). In an example at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of nucleotide bases of abreathable primer are adenine, thymine or uracil or are complementary toadenine, thymine or uracil.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for clonally amplifying anucleic acid template onto a support in an amplification reactionsolution. Optionally, the nucleic acid template is contacted with asupport in a solution comprising a continuous liquid phase. The supportcan include a population of primers, including at least a first primerand a second primer. The population of primers can be immobilized on thesupport, for example by covalent attachment to the support. In someembodiments, the nucleic acid template includes a primer bindingsequence adjacent to a target sequence. The primer binding sequence canby complementary to a sequence of the first primer and optionally asequence of the second primer. The target sequence can benoncomplementary to the primers in the population. In some embodiments,the primer binding sequence of the nucleic acid template is hybridizedto the first primer. The first primer can be extended along the templateusing a polymerase, thereby forming an extended first primer. At least aportion of the primer binding sequence of the template can be separated(e.g., denatured or melted) from the extended first primer. Theseparating is optionally performed while maintaining hybridizationbetween a portion of the template and the extended first primer. Theseparated portion of the primer binding sequence can be subsequentlyhybridized to the second primer. Optionally, such hybridization isperformed while maintaining hybridization between the other portion ofthe template and the extended first primer. The second primer can beextended along the template using a polymerase, thereby forming asupport including an extended first primer and an extended secondprimer. The extended portion of the extended first primer and/or theextended second primer can include sequence complementary to the targetsequence.

In some embodiments, the disclosure relates generally to methods forclonally amplifying a nucleic acid template onto a support in anamplification reaction solution, comprising: contacting a nucleic acidtemplate with a support in a liquid solution, wherein the supportincludes a population of immobilized primers including at least a firstprimer and a second primer, and wherein the nucleic acid templateincludes a primer binding sequence adjacent to a target sequence, wherethe primer binding sequence is complementary to a sequence of the firstprimer and a sequence of the second primer, and the target sequence isnoncomplementary to the primers in the population; hybridizing theprimer binding sequence of the nucleic acid template to the firstprimer; extending the first primer along the template using apolymerase, thereby forming an extended first primer; denaturing atleast a portion of the primer binding sequence of the template from theextended first primer while maintaining hybridization between anotherportion of the template and the extended first primer; hybridizing thedenatured portion of the primer binding sequence to the second primerwhile maintaining hybridization between the other portion of thetemplate and the extended first primer; and extending the second primeralong the template using a polymerase, thereby forming a supportincluding an extended first primer and an extended second primer, wherethe extended first primer and the extended second primer each includesequence complementary to the target sequence. The population of primerscan be comprised of substantially identical primers that differ insequence by no more than one, two, three, four or five nucleotides. Insome embodiments, the primer population is comprised of differentprimers, at least some of which include a sequence that is complementaryto the primer binding sequence of the template. In some embodiments, theprimers of population are noncomplementary to the sequence of the 5′terminal half of the template. In some embodiments, the primers of thepopulation are noncomplementary to the sequence of the 3′ terminal halfof any of the extended primers of the support. In some embodiments, theprimers of the population are noncomplementary to any sequence of thetemplate other than the primer binding sequence.

In some embodiments, the disclosure relates generally to methods forclonally amplifying a population of nucleic acid templates onto apopulation of supports in an amplification reaction solution,comprising: clonally amplifying a first template onto a first nucleicacid template onto a first support according to any of the methodsdisclosed herein, and clonally amplifying a second nucleic acid templateonto a second support according to the same method, wherein bothsupports are included within a single continuous liquid phase during theamplifying.

Among other things, a method is provided of generating a localizedclonal population of immobilized clonal amplicons of a single-strandedtemplate sequence, comprising: (a) attaching the single-strandedtemplate sequence (“template 1”) to an immobilization site (“IS1”),wherein IS1 comprises multiple copies of an immobilized primer (“IS1primer”) which can hybridize substantially to template 1, and template 1is attached to IS1 by hybridization to an IS1 primer, and (b) amplifyingtemplate 1 using IS1 primer and a non-immobilized primer (“SP1 primer”)in solution, wherein amplified strands that are complementary to thesingle-stranded template 1 cannot hybridize substantially whensingle-stranded to primers on IS1, wherein amplification generates alocalized clonal population of immobilized clonal amplicons around thepoint of initial hybridization of template 1 to IS1.

Also provided is a method of generating separated and immobilized clonalpopulations of a first template sequence (“template 1”) and a secondtemplate sequence (“template 2”), comprising amplifying the first andsecond template sequence to generate a population of clonal amplicons oftemplate 1 substantially attached to first immobilization site (“IS1”)and not to a second immobilization site (“IS2”), or a population ofclonal amplicons of template 2 substantially attached to IS2 and not toIS1, wherein: (a) both templates and all amplicons are contained withinthe same continuous liquid phase, where the continuous liquid phase isin contact with a first and second immobilization site (respectively,“IS1” and “IS2”), and where IS1 and IS2 are spatially separated, (b)template 1 when in single-stranded form comprises a first subsequence(“T1-FOR”) at one end, and a second subsequence (“T1-REV”) at itsopposite end, (c) template 2 when in single-stranded form comprises afirst subsequence (“T2-FOR”) at one end, and a second subsequence(“T2-REV”) at its opposite end, (d) IS1 comprises multiple copies of animmobilized nucleic acid primer (“IS1 primer”) that can hybridizesubstantially to T1-FOR and T2-FOR when T1 and T2 are single-stranded,(e) IS2 comprises multiple copies of an immobilized primer (“IS2primer”) that can hybridize substantially to both T1-FOR and T2-FOR whenT1 and T2 are single-stranded, (f) the reverse complement of T1-REV whensingle-stranded cannot hybridize substantially to primers on IS1, butcan hybridize substantially to a non-immobilized primer (“SP1”) in thecontinuous liquid phase; and (g) the reverse complement of T2-REV whensingle-stranded cannot hybridize substantially to primers on IS2, butcan hybridize substantially to a non-immobilized primer (“SP2”) in thecontinuous liquid phase.

Optionally, in any method described herein, any nucleic acid that hasdissociated from one immobilization site is capable of substantiallyhybridizing to both immobilization sites and any movement (e.g.,movement by diffusion, convection) of said dissociated nucleic acid toanother immobilization site is not substantially retarded in thecontinuous liquid phase.

Optionally, in any method described herein, the continuous liquid phaseis in simultaneous contact with IS1 and IS2.

Optionally, in any method described herein, a first portion of atemplate that is bound by an immobilized primer does not overlap with asecond portion of the template whose complement is bound by anon-immobilized primer.

Optionally, in any method described herein, at least one template to beamplified is generated from an input nucleic acid after the nucleic acidis placed in contact with at least one immobilization site.

Optionally, any method described herein comprising the steps of: (a)contacting a support comprising immobilized primers with asingle-stranded nucleic acid template, wherein: hybridizing a firstimmobilized primer to a primer-binding sequence (PB S) on the template(b) extending the hybridized first primer in template-dependentextension to form an extended strand that is complementary to thetemplate and at least partially hybridized to the template; (c)partially denaturing the template from the extended complementary strandsuch that at least a portion of the PBS is in single-stranded form(“free portion”); (d) hybridizing the free portion to a non-extended,immobilized second primer (e) extending the second primer intemplate-dependent extension to form an extended strand that iscomplementary to the template (f) optionally, separating the annealedextended immobilized nucleic acid strands from one another.

Optionally, in any method described herein (a) during amplification,nucleic acid duplexes are formed comprising a starting template and/oramplified strands; which duplexes are not subjected during amplificationto conditions that would cause complete denaturation of a substantialnumber of duplexes.

Optionally, in any method described herein, the single-strandedtemplates are produced by taking a plurality of input double-stranded orsingle-stranded nucleic acid sequences to be amplified (which sequencemay be known or unknown) and appending or creating a first universaladaptor sequence and a second universal adaptor sequence onto the endsof at least one input nucleic acid; wherein said first universal adaptorsequence hybridizes to IS1 primer and/or IS2 primer, and the reversecomplement of said second universal adaptor sequence hybridizes to atleast one non-immobilized primer. The adaptors can be double-stranded orsingle-stranded.

Optionally, in any method described herein, first and second nucleicacid adaptor sequences are provided at first and second ends of saidsingle-stranded template sequence.

Optionally, in any method described herein, a tag is also added to oneor more nucleic acid sequences (e.g., a template or a primer or anamplicons), said tag enabling identification of a nucleic acidcontaining the tag.

Optionally, in any method described herein, all primers on at least oneimmobilization site or support have the same sequence. Optionally, animmobilization site or support comprises a plurality of primers havingat least two different sequences. In some embodiments, an immobilizationsite or support includes at least one target-specific primer.

Optionally, in any method described herein, the continuous medium isflowable. Optionally, intermixing of non-immobilized nucleic acidmolecules is substantially unretarded in the continuous liquid phaseduring at least a portion of the amplification process, e.g., during anyone or more steps or cycles described herein.

Optionally, in any method described herein, intermixing is substantiallyunretarded for a period of time during amplification. For example,intermixing is substantially unretarded during the entire duration ofamplification.

In an embodiment, amplification is achieved using RPA, i.e.,recombinase-polymerase amplification (see, e.g., WO2003072805,incorporated by reference herein). RPA optionally is carried out withoutsubstantial variations in temperature or reagent conditions. In anembodiment herein, partial denaturation and/or amplification, includingany one or more steps or methods described herein, can be achieved usinga recombinase and/or single-stranded binding protein. Suitablerecombinases include RecA and its prokaryotic or eukaryotic homologues,or functional fragments or variants thereof, optionally in combinationwith single-strand binding proteins (SSBs). In an embodiment, therecombinase agent optionally coats single-stranded DNA (ssDNA) such asan amplification primer to form a nucleoprotein filament strand whichinvades a double-stranded region of homology on a template. Thisoptionally creates a short hybrid and a displaced strand bubble known asa D-loop. In an embodiment, the free 3′-end of the filament strand inthe D-loop is extended by DNA polymerases to synthesize a newcomplementary strand. The complementary strand displaces theoriginally-paired partner strand of the template as it elongates. In anembodiment, one or more of a pair of amplification primers are contactedwith one or more recombinase agents before be contacted with a templatewhich is optionally double-stranded.

In any method described herein, amplification of a template (targetsequence) comprises contacting a recombinase agent with one or more ofat least one pair of amplification primers, thereby forming one or more“forward” and/or “reverse” RPA primers. Any recombinase agent that hasnot associated with the one or more primers is optionally removed.Optionally, one or more forward RPA primers are then contacted with atemplate strand, which optionally has a region of complementarity to atleast one RPA primer. The template strand can be hybridized to aContacting of a RPA primer with a complementary template optionallyresults hybridization between said primer and the template. Optionally,the 3′ end of the primer is extended along the template with one or morepolymerases (e.g., in the presence of dNTPs) to generate a doublestranded nucleic acid and a displaced template strand. The amplificationreaction can comprise repeated cycles of such contacting and extendinguntil a desired degree of amplification is achievable. Optionally thedisplaced strand of nucleic acid is amplified by a concurrent RPAreaction. Optionally, the displaced strand of nucleic acid is amplifiedby contacting it in turn with one or more complementary primers; and (b)extending the complementary primer by any strategy described herein.

In an embodiment the one or more primers comprise a “forward” primer anda “reverse” primer. Placing both primers and the template in contactoptionally results in a first double stranded structure at a firstportion of said first strand and a double stranded structure at a secondportion of said second strand. Optionally, the 3′ end of the forwardand/or reverse primer is extended with one or more polymerases togenerate a first and second double stranded nucleic acid and a first andsecond displaced strand of nucleic acid. Optionally the second displacedstrand is at least partially complementary to each other and canhybridize to form a daughter double stranded nucleic acid which canserve as double stranded template nucleic acid in a subsequentamplification cycles.

Optionally said first and said second displaced strands is at leastpartially complementary to said first or said second primer and canhybridize to said first or said second primer.

In an alternative embodiment of any method or step or composition orarray described herein, the support optionally comprises immobilizedprimers of more than one sequence. After a template nucleic acid strandhybridizes to first complementary immobilized primer, the first primercan then be extended and the template and the primer can be separatedpartially or completely from one another. The extended primer can thenbe annealed to a second immobilised primer that has different sequencefrom the first, and the second primer can be extended. Both extendedprimers can then be separated (e.g., fully or partially denatured fromone another) and can be used in turn as templates for extension ofadditional immobilized primers. The process can be repeated to provideamplified, immobilised nucleic acid molecules. In an embodiment, thisamplification results in immobilized primer extension products of twodifferent sequences that are complementary to each other, where allprimer extension products are immobilized at the 5′ end to the support

In some embodiments, the disclosed methods include amplifying, whereinthe amplifying includes strand flipping. In a “flipping” embodimentdescribed below, two or more primers are extended to form two or morecorresponding extended strands. Optionally, the two or more primers thatare extended comprise or consist essentially of substantially identicalsequence, and the extended portions of corresponding extended strand areat least partly non-identical and/or complementary to each other.

One exemplary embodiment of flipping is as follows. A starting templateis amplified, e.g., by template walking, to generate a plurality ofprimer-extended strands (which for convenience will be designated as“forward” strands). Optionally, the forward strands are complementary tothe starting template. Optionally, the forward strands are immobilizedon the support. Optionally, the forward strands comprise substantiallyidentical sequence, e.g., the forward strands are substantiallyidentical to each other. In an embodiment the forward strands are formedby extension of one or more primers immobilized on a support (“forward”primers). The forward primers and/or the forward strands are optionallyattached to the support at or near their 5′ ends. Optionally, one ormore of the primer-extended forward strands comprises a 3′ sequence(called a self-hybridizing sequence) that is absent in the unextendedprimer and can hybridize under the conditions of choice to a 5′ sequence(this process will be termed “self-hybridization”). The 5′ sequence isoptionally part of the unextended forward primer. In an example, theforward extension product forms a “stem-loop” structure upon suchhybridization. Optionally, the unextended forward primer comprises a“cleavable” nucleotide at or near its 3′ terminus that is susceptible tocleavage. In an embodiment, the cleavable nucleotide is linked to atleast one other nucleotide by a “scissile” internucleoside linkage thatcan be cleaved under conditions that will not substantially cleavephosphodiester bonds.

After extension, the forward-primer extension product (i.e., the forwardstrand) is optionally allowed to self-hybridize. In a furtherembodiment, after allowing for self-hybridization the forward strand iscleaved at a scissile linkage of a cleavable nucleotide (for example anucleotide which forms a scissile linkage with a neighboringnucleotide). The cleavage results in two fragments of theprimer-extension product (i.e., the extended forward strand). In anembodiment, a first fragment comprises at least a portion of theoriginal unextended forward primer. Optionally, the first fragment doesnot comprise any extended sequence. Optionally, the first fragment isimmobilized (e.g., because the unextended forward primer was alreadyimmobilized). In an embodiment, a second fragment comprises extendedsequence. Optionally the second fragment comprises any 3′ portion of theunextended primer beyond the cleavable nucleotide or does not compriseany portion of the unextended primer. Optionally, the second fragment ishybridized to the first portion through its self-hybridizing sequence.

In an example, the cleavable nucleotide is one that is removed by one ormore enzymes. The enzyme can for instance be a glycosylase. Theglycosylase optionally has N-glycosylase activity which releases thecleavable nucleotide from double stranded DNA. Optionally, the removalof the cleavable nucleotide generates an abasic, apurinic orapyrimidinic site. The abasic site can optionally be further modified,for example by another enzymatic activity. Optionally, the abasic siteis modified by a lyase to generate a base gap. The lyase for examplecleaves 3′ and/or 5′ to the abasic site. Cleavage optionally occurs atboth the 5′ and 3′ end by the lyase, resulting in removing the abasicsite and leaving a base gap. Exemplary cleavable nucleotides such as5-hydroxy-uracil, 7, 8-dihydro-8-oxoguanine (8-oxoguanine),8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxinB1-fapy-guanine, 5-hydroxy-cytosine can be recognized and removed byvarious glycosylases to form an apurinic site. One suitable enzyme isformamidopyrimidine [fapy]-DNA glycosylase, also known as 8-oxoguanineDNA glycosylase or FPG. FPG acts both as a N-glycosylase and anAP-lyase. The N-glycosylase activity optionally releases damaged purinesfrom double stranded DNA, generating an apurinic (AP site), where thephosphodiester backbone is optionally intact. The AP-lyase activitycleaves both 3′ and 5′ to the AP site thereby removing the AP site andleaving a one-base gap. In an example the cleavable nucleotide is8-oxoadenine, which is converted to a one-base gap by FPG with bothglycosylase and lyase activities.

In another embodiment the cleavable nucleotide is uridine. Optionally,the uridine is cleaved by “USER” reagent, which includes Uracil DNAglycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII, whereUDG catalyses the excision of a uracil base, forming an abasic(apyrimidinic) site while leaving the phosphodiester backbone intact,and where the lyase activity of Endonuclease VIII breaks thephosphodiester backbone at the 3′ and 5′ sides of the abasic site sothat base-free deoxyribose is released, after which kinase is optionallyused to convert the phosphate group on the 3′ end of cleaved product toan —OH group).

At least one cleaved fragment is optionally contacted with a polymerase.Optionally the first immobilized fragment can be extended by thepolymerase. If so desired the second hybridized fragment can act astemplate for extension of the first fragment. In an embodiment a“flipped” double-stranded extension product is formed. This flippedproduct can optionally be subjected to template walking in any mannerdescribed herein. When both flipped and unflipped are subjected totemplate walking, a cluster of two different extension products isformed, where both extension products have an identical portion(corresponding to the unextended primers) and portion complementary toeach other, corresponding to the extended portions of the extensionproducts.

In an embodiment, a sequence of interest, such as a self-hybridizingsequence or a new primer-binding site, can be optionally be added at the3′ ends of extended forward strands by contacting the extended forwardstrands with a single-stranded “splice” adaptor sequence in the presenceof extension reagents (e.g., a polymerase and dNTPs). This splicesequence optionally comprises a 3′ portion that is substantiallycomplementary to a 3′ end portion of the extended forward strand, and a5′ portion that is substantially complementary to the sequence ofinterest to be added. After hybridizing the splice adaptor to the 3′ endof the extended forward strand, the forward strand is subjected totemplate-dependent polymerase extension using the splice adaptor astemplate. Such extension results in the addition of the sequence ofinterest to the 3′ end of the extended forward strand.

Thus, any method of primer extension and/or amplification describedherein can include any one or more of the following steps: (a) extensionof immobilized forward primers by template walking to generate aplurality of extended forward strands which are optionally identical;(b) optionally hybridizing a splice adaptor to a 3′ end of the extendedforward strands and subjecting the forward strands to template-dependentextension using the splice adaptor as a template, thereby adding afurther 3′ sequence to the further-extended forward strands, wherein aportion of the added 3′ sequence is complementary to a portion of theunextended forward primer and hybridizes thereto to form a stem-loopstructure; (c) cleaving the forward strands at a scissile linkage of acleavable nucleotide located at or near the junction of unextendedforward primer sequence and extended forward strand sequence; andoptionally removing the cleavable nucleotide, thereby generating twocleaved fragments, the first fragment comprising a portion of anunextended forward primer hybridized to a 3′ primer-complementarysequence on the second fragment; (d) optionally subjecting the firstfragment to polymerase extension using the second fragment as templateto generate a flipped forward strand; (e) optionally hybridizing asecond splice adaptor to a 3′ end of the flipped forward strand, andsubjecting the forward strands to template-dependent extension using thesplice adaptor as a template, thereby adding a further 3′ sequence tothe flipped forward strands, wherein a portion of the added 3′ sequenceis a new primer-binding sequence that is absent in the flipped strands;(f) selectively extending or amplifying the flipped strands whichcomprise the new primer-binding sequence by contacting with the newprimer and extending or amplifying by any method, e.g., as describedherein. The new primer will not bind to unflipped strands or to flippedstrands that were not further extended in step (e).

FIGS. 8A-C show a schematic depiction of an exemplary strand-flippingand walking strategy. FIG. 8A Template walking, FIG. 8B Strand flippingto generate flipped strands, FIG. 8C addition of new primer-bindingsequence Pg′ on final flipped strands.

Optionally, a single support is used in any of the amplification methodsherein, where the single support has a plurality of primers that canhybridize to the templates. In such an embodiment, the concentration ofthe template collection is adjusted before it is contacted with a solidsupport so that individual template molecules in the collection getattached or associated (e.g., by hybridization to primers immobilized onthe solid support) at a density of at least 10², 10³, 10⁴, 10⁵, 4×10⁵,5×10⁵, 6×10⁵, 8×10⁵, 10⁶, 5×10⁶ or 10⁷ molecules per mm².

Optionally, individual template molecules are amplified in-situ on thesupport, giving rise to clonal populations that are spatially-centeredaround the point of hybridization of the initial template. Optionally,the amplification generates no more than about 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹⁵, or 10²⁰ amplicons from a singleamplified template. Optionally, the colonies of clonal amplicons aresituated on the solid support at a density of at least 10², 10³, 10⁴,10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 8×10⁵, 10⁶, 5×10⁶ or 10⁷ molecules per mm².

In some embodiments, the nucleic acid collection can be contacted withone or more supports under conditions where multiple nucleic acids bindto the same support. Such contacting can be particularly useful inmethods that involve parallel clonal amplification of nucleic acids indifferent regions of the same support. The ratio of the number ofnucleic acids to the surface area of the support can be adjusted tofacilitate mono-clone formation by, e.g., ensuring that the nucleicacids are appropriately spaced in the support to favor formation ofmonoclonal populations of amplified nucleic acids without substantialcross-contamination between different clonal populations. For examplewhere a single support us used, the collection of nucleic acids to beamplified is adjusted to such a dilution that the resulting amplifiedclonal populations generated from individual nucleic acids are generallydiscrete or distinct, e.g., without overlap. For example, individualnucleic acids within 50%, 70%, 80% or 90% or more of the amplifiedclonal populations are not interspersed with substantially non-identicalnucleic acids. Optionally, different amplified populations are not incontact or completely overlapping with other amplified populations, orare distinguishable from each other using a detection method of choice.

In some embodiments, the nucleic acids are attached to the surface of asupport. In some embodiments, the nucleic acids can be attached withinthe support. For example, for supports comprised of hydrogel or otherporous matrices, the nucleic acids can be attached throughout the volumeof the support including on the surface and within the support.

In some embodiments, the support (or at least one support in apopulation of supports) can be attached to at least one primer,optionally to a population of primers. For example, the support (or atleast one support) can include a population of primers. The primers ofthe population can be substantially identical each other, or may includea substantially identical sequence. One, some or all of the primers caninclude a sequence that is complementary to a sequence within one ormore nucleic acid templates. In some embodiments, the population ofprimers can include at least two noncomplementary primers.

The primers can be attached to the support through their 5′ end, andhave free 3′ ends. The support can be the surface of a slide or thesurface of a bead. The primers have low melting temperature, such asoligo (dT)₂₀, and can hybridize to the low T_(m) region of thecollection adaptor. The distances between the primers need to be shorterthan the adapter length to allow templates waking, or alternatively, along primer with 5′ end long linker will increase the chance of walking.

In some embodiments, the support is attached to and/or contacted with aprimer and a template (or reverse strand) under conditions where theprimer and template hybridize to each other to form a nucleic acidduplex. The duplex can include a double stranded portion that comprisescomplementary sequences of the template and primer, where at least onenucleotide residue of the complementary sequences are base paired witheach other. In some embodiments, the duplex can also include a singlestranded portion. The duplex can also include a single stranded portion.The single stranded portion can include any sequence within the template(or primer) that is not complementary to any other sequence in theprimer (or template).

A non-limiting exemplary method of clonal nucleic acid amplification ona support is as follows. A nucleic acid (which shall be designated forconvenience as the reverse strand) is clonally amplified onto a supporton which multiple copies of a complementary forward primer are attached.An exemplary nucleic acid is one of a plurality of DNA collectionmolecules, that for example the plurality of nucleic acid members haveone or more common (“adaptor”) sequences at or near their 5′ and/or 3′ends and variable sequences in between, such as gDNA or cDNA. In anembodiment, the 3′ common portion, e.g., adaptor, has a breathable(e.g., low T_(m)) region, and the 5′ common sequence (e.g., adaptor)optionally has a less breathable (e.g., higher T_(m)) region, or viceversa. In another embodiment, both the 5′ and 3′ common sequences arebreathable. The breathable (e.g., low T_(m)) region is for example aregion that is rich in A, T and/or U, such as an AT (or U)-richsequence, such as polyT, polyA, polyU and any combinations of A, T and Ubases, or bases complementary to such bases. Exemplary methods aredescribed herein.

One non-limiting exemplary method of clonal nucleic acid amplificationon a support via “template walking” is shown in FIG. 1 . A non-limitingdescription of an exemplary method of template walking is as follows.

A double stranded DNA library molecule is denatured and the singlestranded DNA is attached to the support through hybridization to theprimers on the surface. The ratio of number of DNA molecules to supportarea or number of beads is set to facilitate mono-clone formation.

Primers are attached on a support through their 5′ and have free 3′. Thesupport can be the surface of a slide or the surface of a bead. Theprimers have low melting temperature, such as oligo (dT)₂₀ or oligo(dA)₃₀ and can hybridize to the low Tm region of the library adaptor.The distances between the primers can be shorter than the adapter lengthto allow templates waking, or alternatively, a long primer with 5′ endlong linker will increase the chance of walking.

A nucleic acid is clonally amplified onto a support on which multiplecopies of a primer are attached. An exemplary nucleic acid is one of aplurality of DNA library molecules, which for example have one or morecommon (e.g., “adaptor”) sequences at their 5′ and/or 3′ ends andvariable sequences in between, such as gDNA or cDNA. In an embodiment,the 3′ adaptor has a low Tm region, and the 5′ adaptor optionally has ahigher Tm region, or vice versa. The low Tm region is for example apyrimidine-rich region, such as an AT (or U)-rich sequence, such aspolyT, polyA, polyU and any combinations of A, T and U bases or basescomplementary to such bases. Exemplary methods are described herein.

One or more primers, whether in soluble form or attached to a support,is incubated with a DNA polymerization or extension reaction mix, whichoptionally comprises any one or more of reagents such as enzyme, dNTPsand buffers. The primer (e.g., a forward primer) is extended.Optionally, the extension is a template-dependent extension of a primeralong a template comprising the successive incorporation of nucleotidesthat are individually complementary to successive nucleotides on thetemplate, such that the extended or nonextended forward primer iscomplementary to the reverse strand (also termed antiparallel orcomplementary). Optionally, the extension is achieved by an enzyme withpolymerase activity or other extension activity, such as a polymerase.The enzyme can optionally have other activities including 3′-5′exonuclease activity (proofreading activity) and/or 5′-3′ exonucleaseactivity. Alternatively, in some embodiments the enzyme can lack one ormore of these activities. In an embodiment the polymerase hasstrand-displacing activity. Examples of useful strand-displacingpolymerases include Bacteriophage Φ29 DNA polymerase and Bst DNApolymerase. Optionally, the enzyme is active at elevated temperatures,e.g., at or above 45° C., above 50° C., 60° C., 65° C., 70° C., 75° C.,or 85° C.

An exemplary polymerase is Bst DNA Polymerase (Exonuclease Minus), is a67 kDa Bacillus stearothermophilus DNA Polymerase protein (largefragment), exemplified in accession number 2BDP_A, which has 5′-3′polymerase activity and strand displacement activity but lacks 3′-5′exonuclease activity. Other polymerases include Taq DNA polymerase Ifrom Thermus aquaticus (exemplified by accession number 1TAQ), Eco DNApolymerase I from Escherichia coli (accession number P00582), Aea DNApolymerase I from Aquifex aeolicus (accession number 067779), orfunctional fragments or variants thereof, e.g., with at least 80%, 85%,90%, 95% or 99% sequence identity at the nucleotide level.

Generally, the extension step produces a nucleic acid, which comprises adouble-stranded duplex portion in which two complementary strands arehybridized to each other. In one embodiment, walking involves subjectingthe nucleic acid to partially-denaturing conditions that denature aportion of the nucleic acid strand but are insufficient to fullydenature the nucleic acid across its entire length. In an embodiment,the nucleic acid is not subjected to fully-denaturing conditions duringa portion or the entire duration of the walking procedure.

In an embodiment, the sequence of the negative and/or positive stranddesigned such that a primer-binding sequence or a portion thereof isbreathable, i.e., is susceptible to denaturation under the conditions ofchoice (e.g., amplification conditions). The breathable portion isoptionally more susceptible than a majority of nucleic acids of similarlength with randomized sequence, or more susceptible than at leastanother portion of the strand comprising the breathable sequence.Optionally, the breathable sequence shows a significant amount ofdenaturation (e.g., at least 10%, 20%, 30%, 50%, 70%, 80%, 90% or 95% ofmolecules are completely denatured across the breathable sequence) atthe amplification conditions of choice. For example the breathablesequence is designed to be fully-denatured in 50% of strand molecules at30, 35, 40, 42, 45, 50, 55, 60, 65 or 70° C. under the conditions ofchoice (e.g., amplification conditions).

Where partial denaturation is achieved by heating or elevatedtemperatures, an exemplary breathable PBS may be pyrimidine-rich (e.g.,with a high content of As and/or Ts and/or Us). The PBS comprises forexample a poly-A, poly-T or poly-U sequence, or a polypyrimidine tract.One or more amplification or other primers (e.g., an immobilized primer)are optionally designed to be correspondingly complementary to theseprimer-binding sequences. An exemplary PBS of a nucleic acid strandcomprises a poly-T sequence, e.g., a stretch of at least 10, 15, 20, 25or 30 thymidine nucleotides, while the corresponding primer has acomplementary sequence to the PBS, e.g., a stretch of at least 10, 15,20, 25 or 30 adenosine nucleotides. Exemplary low-melt primersoptionally have a high proportion (e.g., at least 50%, 60%, 65%, 70%,75%, 80%, 85% 90% 95% or 100%) of nucleobases that generally (e.g.,under amplification conditions of choice) form no more than two hydrogenbonds with a complementary base when the primer is hybridized to acomplementary template. Examples of such nucleobases include A(adenine), T (thymine) and U (uracil). Exemplary low-melt primersoptionally have a high proportion of any one or more of A (adenine), T(thymine) and/or U (uracil) nucleotides or derivatives thereof. In anembodiment, the derivatives comprise nucleobases that are complementaryto A (adenine), T (thymine) and/or U (uracil). The portion of the primerthat hybridizes to the PBS optionally has at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90% 95% or 100% of A (adenine), T (thymine) or U (uracil)nucleotides, or any combination thereof. In another example, the portionof the primer that hybridizes to the PBS comprises a polyA sequence(e.g., at least 5, 10, 15, 20, 25 or 30 nucleotides long). Otherexemplary primers comprise (NA_(x))_(n) repeats. Optionally, n (in lowercase) is from 2 to 30, e.g., from 3 to 10, for example 4 to 8. “N” (inupper case) is any nucleotide—and optionally, N is C or G. “A” is theshorthand convention for adenine, and “x” denotes the number of adenineresidues in the repeat, for example 2, 3, 4, 5, 6, 10 or more. Exemplaryprimers comprise multiple repeats of (CAA)_(n), CA)_(n), (CAAA)_(n) oreven (GAA)_(n).

Optionally only one strand (e.g., the forward or the reverse strand) hasa breathable PBS. In another embodiment, both the forward and reversestrands have a breathable PBS. The breathable PBS is optionallycomplementary to a primer that is either immobilized to a support or isnot immobilized (e.g., in soluble form). Optionally the strandcomprising the breathable PBS is either immobilized to a support or isnot immobilized (e.g., in soluble form). Optionally both primers areimmobilized, or both strands are immobilized. Optionally neither primeris immobilized, or neither strand is immobilized.

An amplification cycle optionally comprises breathing, annealing andextension. The nucleic acid to be amplified is optionally subjected toconditions which are suitable for or optimized for at least one of thesesteps. In an embodiment, the nucleic acid is subjected to conditionswhich are suitable for more than one of these steps, (e.g., annealingand extension, or breathing and extension). In some instances, all threeof these steps can take place simultaneously under the same conditions.

In an exemplary method the nucleic acid can be subjected to conditionswhich permit or facilitate breathing. In an embodiment, “breathing” issaid to occur when the two strands of a double-stranded duplex aresubstantially hybridized to each other, but are denatured across a localportion of interest (e.g., the terminal ends or primer-binding sites).One or more breathable sequences (e.g., a forward and/or reverse PBShaving a low Tm portion) of the nucleic acid gets locally denatured(“breathes”) from a first complementary strand (e.g., a forward orreverse strand) which it is hybridized to, and is thus made available tohybridize to another second strand. An exemplary first strand is aprimer extension product from a first primer. An exemplary second strandis for example a second unextended primer (e.g., a PBS-complementaryoligonucleotide comprising, e.g., a dT or dA sequence). Optionally, thefirst and second strands are immobilized on a support, and can beclosely situated (e.g., in close enough proximity to allow walking). Theconditions for breathing are optionally partially-denaturing conditionsunder which the PBS is generally denatured but another portion ofnucleic acid remains in a hybridized or double-stranded state.Optionally, DNA helicase can be included in the reaction mix tofacilitate the partial denaturing.

Optionally, the nucleic acid is then subjected to conditions whichfacilitate annealing, e.g., the temperature is decreased, to enablehybridization between the breathable PBS and the second strand. In anembodiment, the same conditions are used to facilitate both breathingand extension. In another embodiment, annealing conditions are differentfrom breathing conditions—for example, the annealing conditions arenondenaturing conditions or conditions that favor denaturation less thanthe breathing conditions. In an example, annealing conditions involve alower temperature (such as 37° C.) than breathing conditions, in which ahigher temperature (e.g., 60-65° C.) is used. Optionally, fullydenaturing conditions are avoided during one or more cycles ofamplification (e.g., the majority of amplification cycles orsubstantially all amplification cycles).

Optionally, one or more of the PBS-breathing and primer extension stepsare repeated multiple times to amplify an initial nucleic acid. Whereone or more nucleic acid reagents (e.g., primers) are immobilized to asupport, the primer-extension products remain substantially attached tothe support, e.g., by virtue of attachment of an unextended extendedprimer to the support prior to amplification, or by hybridization tosuch a primer).

Optionally, a sample is prepared of a population of one or more nucleicacids to be amplified. The population of nucleic acids can be insingle-stranded or double-stranded form; optionally one or more nucleicacids individually comprises a nucleic acid strand with a known 3′ endsequence and a known 5′ end sequence which are substantially identicalor complementary to the one or more primers used in the amplification. A3′ portion of the nucleic acid strand can for example be complementaryto an immobilized primer, whereas a 5′ portion can be identical to asoluble primer. The 5′ and/or 3′ portions can be common (“universal”) orinvariant between individual nucleic acids within the population.Optionally, the nucleic acids within the population individuallycomprise variant (e.g., unknown) sequence between the common portions,such as genomic DNA, cDNAs, mRNAs, mate-pair fragments, exomes. etc. Thecollection can for example have enough members to ensure over 50%, 70%,or 90% coverage of the corresponding genetic source (e.g., the genome orthe exome).

In some embodiments, the disclosure relates generally to compositions,as well as related systems, apparatuses, kits and methods, for nucleicacid amplification, comprising a reaction mixture for nucleic acidamplification.

In some embodiments, the disclosure relates generally to compositions,as well as related systems, apparatuses, kits and methods, for nucleicacid amplification, comprising a reaction mixture including a continuousliquid phase, the continuous liquid phase containing (i) a polymeraseand (ii) a plurality of supports, at least one of the supports beingattached to a substantially monoclonal population of nucleic acids.

In some embodiments, the disclosure relates generally to compositions,as well as related systems, apparatuses, kits and methods, for nucleicacid amplification, comprising a reaction mixture including a continuousliquid phase, the continuous liquid phase containing (i) a recombinaseand (ii) a plurality of supports including a first support and a secondsupport.

In some embodiments, the disclosure relates generally to compositions(as well as related systems, apparatuses, kits and methods) for nucleicacid amplification, comprising: a reaction mixture including acontinuous liquid phase, the continuous liquid phase containing (i) aplurality of supports including a first support and a second support,(ii) a plurality of different polynucleotides including a firstpolynucleotide and a second polynucleotide and (iii) reagents forisothermal nucleic acid amplification. In some embodiments, the reagentsfor nucleic acid amplification include a polymerase and one or moretypes of nucleotide (e.g., a plurality of nucleotides). Optionally, thereagents for isothermal nucleic acid amplification include arecombinase.

Optionally, the first and second polynucleotides have differentsequences.

Optionally, at least one end of at least one of the plurality ofdifferent polynucleotides is joined to at least one oligonucleotideadaptor.

Optionally, at least one end of at least some of the plurality ofdifferent polynucleotides includes a common sequence.

Optionally, at least two of the different polynucleotides in thereaction mixture include a common sequence.

Optionally, the first and second polynucleotides are different.

In some embodiments, the liquid phase includes one or more supports ofthe plurality include a primer.

In some embodiments, the disclosure relates generally to compositions,as well as related systems, apparatuses, kits and methods, for nucleicacid amplification, comprising a reaction mixture for nucleic acidamplification.

Optionally, the reaction mixture includes a continuous liquid phase.

Optionally, the reaction mixture can be used to conduct isothermal orthermocycling nucleic acid amplification.

Optionally, the continuous liquid phase includes any one or anycombination of (i) one or more polymerases and/or (ii) at least onesupport.

Optionally, the continuous liquid phase includes a plurality ofsupports.

Optionally, the continuous liquid phase includes a first support.

Optionally, the continuous liquid phase includes a second support.

Optionally, at least one of the supports in the plurality can beattached to a substantially monoclonal population of nucleic acids.

Optionally, the first support can be attached to a first substantiallymonoclonal population of nucleic acids.

Optionally, the second support can be attached to a second substantiallymonoclonal population of nucleic acids.

Optionally, the first and the second substantially monoclonal populationof nucleic acids comprise different sequences or essentially identicalsequences.

Optionally, the first and second substantially monoclonal populations ofnucleic acids do or do not hybridize with each other under stringenthybridization conditions.

Optionally, the first and second substantially monoclonal populations ofnucleic acids are non-identical.

Optionally, the first and second substantially monoclonal populations ofnucleic acids are non-complementary.

Optionally, the reaction mixture includes nucleotides that are notextrinsically labeled. For example, the nucleotides can be naturallyoccurring nucleotides, or synthetic analogs that do not includefluorescent moieties, dyes, or other extrinsic optically detectablelabels.

Optionally, the reaction mixture includes nucleotides that are naturallyoccurring nucleotides. Optionally, the nucleotides do not include groupsthat terminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like).

Optionally, the reaction mixture is contained in a single reactionvessel.

Optionally, the reaction mixture comprises an isothermal or athermocycling reaction mixture.

Optionally, the plurality of supports comprises beads, particles,microparticles, spheres, gels, filters or inner walls of a tube.

Optionally, at least one of the support in the plurality can be attachedto a plurality of nucleic acids.

Optionally, at least one of the support in the plurality can be attachedto one or more primers. The primers may be the same (or include a commonsequence), or different.

Optionally, at least one of the supports can be attached to a pluralityof a first primer.

Optionally, at least one of the supports is attached to a plurality of afirst primer and to a plurality of a second primer.

Optionally, the plurality of the first primer comprises essentiallyidentical sequences.

Optionally, the plurality of the first primer includes at least onefirst primer containing a sequence that is identical, or complementary,to at least a portion of a polynucleotide of the plurality of differentpolynucleotides.

Optionally, the plurality of the second primer includes at least onesecond primer containing a sequence that is identical, or complementary,to at least a portion of a polynucleotide of the plurality of differentpolynucleotides.

In some embodiments, at least one polynucleotide of the plurality ofdifferent polynucleotides includes a first sequence that issubstantially identical or substantially complementary to a sequencewithin the first primer. In some embodiments, the at least onepolynucleotide also includes a second sequence that is substantiallyidentical or substantially complementary to a sequence within the secondprimer. In some embodiments, substantially all of the polynucleotides inthe plurality of different polynucleotides including the first sequenceand the second sequence.

Optionally, at least one of the supports in the plurality is attached toa plurality of 2-10 different primers.

Optionally, the plurality of the 2-10 different primers comprisesdifferent sequences.

Optionally, the plurality of the 2-10 different primers comprises atleast one sequence that hybridizes with at least a portion of thedifferent polynucleotides.

Optionally, the plurality of the 2-10 different primers comprise atleast one sequence that hybridizes with at least a portion of a commonsequence in the different polynucleotides.

Optionally, at least one of the supports is attached to at least oneuniquely identifying barcode sequence.

Optionally, the first and second substantially monoclonal population ofnucleic acids have sequences that are essentially the same or aredifferent.

Optionally, the reaction mixture includes at least one recombinase.

Optionally, the recombinase can catalyze homologous recombination,strand invasion and/or D-loop formation.

Optionally, the recombinase is part of a nucleoprotein filament thatincludes the recombinase bound to a primer attached to a support in thereaction mixture. The primer bound by the recombinase can be attached toa support or in solution.

Optionally, the reaction mixture includes a nucleoprotein complex, or aplurality of nucleoprotein complexes.

Optionally, the reaction mixture includes a first nucleoprotein complex.

Optionally, the reaction mixture includes a second nucleoproteincomplex.

Optionally, at least one nucleoprotein complex of the plurality includesat least one recombinase bound to a primer.

Optionally, the reaction mixture includes a first nucleoprotein complexwhich includes at least one recombinase bound to a first primer.

Optionally, the reaction mixture includes a second nucleoprotein complexwhich includes at least one recombinase bound to a second primer.

Optionally, the recombinase comprises a phage recombinase from T4, T2,T6, Rb69, AehI, KVP40, Acinetobacter phage 133, Aeromonas phage 65,cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14; Rb32,Aeromonas phage Vibrio phage nt−1, phi-1, Rb16, Rb43, Phage 31, phage44RR2.8t, Rb49, phage Rb3, or phage LZ2.

Optionally, the recombinase comprises a uvsX recombinase from T4bacteriophage or a recA recombinase from E. coli.

Optionally, the reaction mixture further includes a polymerase.

Optionally, the polymerase lacks a 5′ to 3′ exonuclease activity.

Optionally, the polymerase includes a strand displacing activity.

Optionally, the polymerase comprises a thermostable or thermo-sensitivepolymerase.

Optionally, the polymerase comprises a DNA polymerase or an RNApolymerase.

Optionally, the reaction mixture further includes at least one type ofnucleotide.

Optionally, the reaction mixture includes nucleotides that are notextrinsically labeled. For example, the nucleotides can be naturallyoccurring nucleotides, or synthetic analogs that do not includefluorescent moieties, dyes, or other extrinsic optically detectablelabels.

Optionally, the reaction mixture includes nucleotides that are naturallyoccurring nucleotides. Optionally, the nucleotides do not include groupsthat terminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like).

Optionally, at least one support of the plurality includes at least oneprimer.

Optionally, the first support is attached to a first substantiallymonoclonal nucleic acid population and the second support is attached toa second substantially monoclonal nucleic acid population.

Optionally, the first and second substantially monoclonal nucleic acidpopulations have different nucleic acid sequences.

Optionally, the first and second substantially monoclonal nucleic acidpopulations do not hybridize with each other under stringenthybridization conditions.

Optionally, the first and second nucleic acid substantially monoclonalnucleic acid populations are non-identical and non-complementary.

Optionally, the reaction mixture includes (i) at least twopolynucleotide templates to be amplified and/or (ii) at least onenucleoprotein filament complex.

Optionally, the reaction mixture includes at least one polynucleotide,primer, template, or amplification product that is attached to a dragcompound. The term “drag compound” and its variants, as used herein,describe any chemical compositions that can be attached to nucleic acidsand retard their diffusion through a reaction mixture, but still permitnucleic acid synthesis to proceed using such polynucleotide, primer,template or amplification product in a nucleic acid synthesis reaction.Attachment of such drag compounds to nucleic acids within a synthesisreaction typically reduces the mobility of such nucleic acids in thereaction mixture and can be useful in preventing cross-contamination ofamplification products or templates between different syntheticreactions occurring with the same reaction mixture. In some embodiments,the attachment of drag components to one or more nucleic acid componentscan increase the number or proportion of monoclonal products.

In some embodiments, the present teachings provide methods for nucleicacid amplification comprising at least one mobility-altered nucleic acid(e.g., a primer). In some embodiments, a mobility-altered nucleic acidexhibits increased or decreased mobility through an aqueous medium. Insome embodiments, a modified nucleic acid comprises a nucleic acid(e.g., a primer) attached at any location along the nucleic acid lengthto one or more compounds (e.g., drag compound) that alter the mobilityof a nucleic acid through an aqueous medium. In some embodiments, a dragcompound that alters the mobility of a nucleic acid can be attached toany primer in a nucleic acid amplification reaction, including a first,second, third, fourth, or any other primer. For example, one or moredrag compounds can be attached to a nucleic acid at any one or anycombination of the 5′ end, the 3′ end, and/or an internal location. Insome embodiments, a modified nucleic acid can be attached covalently ornon-covalently to a drag compound that changes the mobility of thenucleic acid through an aqueous medium. For example, a drag compound canprovide hydro hydrodynamic drag when attached to a nucleic acid byaltering the overall size, length, radius, shape or electrical charge ofthe modified nucleic acid compared to the nucleic acid lacking theattached compound. In some embodiments, a drag compound attached to anucleic acid can alter interaction between the nucleic acid and anaqueous medium compared to the interaction between the aqueous mediumand a nucleic acid lacking the attached compound. In some embodiments,the drag compound can be synthetic, recombinant or naturally-occurring.In some embodiments, the drag compound can be charged, uncharged, polaror hydrophobic. In some embodiments, the drag compound can be linear,branched or have a dendrimeric structure. In some embodiments, the dragcompound can comprise a single moiety or polymers of nucleosides,saccharides, lipids, or amino acids.

Optionally, a drag compound comprises a saccharide moiety, apolysaccharide, a protein, a glycoprotein or polypeptide. Optionally, adrag compound comprises BSA, lysozyme, beta-actin, myosin, lactalbumin,ovalbumin, beta-galactosidase, lactate dehydrogenase or immunoglobulin(e.g., IgG).

Optionally, a drag compound that alters the mobility of the nucleic acidthrough an aqueous medium comprises one or more polyethylene oxide (PEO)or polypropylene oxide (PPO) moieties, including polymers ofpolyethylene oxide (PEO) or polypropylene oxide (PPO). Non-limitingexamples of such polymers include triblock copolymers (e.g.,PEO-PPO-PEO), Pluronics™-type polymers, and hydrophobically-modified PEOpolymers. Optionally, the drag compound comprises one or more amino acidmoieties, polypeptides and polypeptides. Optionally, the drag compoundcomprises a saccharide moiety, polysaccharides, hydrophobically-modifiedpolysaccharides, cellulose derivatives, sodium carboxymethylcellulose,hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,or hydroxypropylmethyl cellulose. Optionally, the drag compoundcomprises a hydrophobically-modified alkali-soluble associative (HASE)polymers, hydrophobically modified polyacrylamides, thermally responsivepolymers, or N-isopropylacrylamide (NTPAAm), Optionally, the dragcompound comprises a poly(ethylene glycol) methylether acrylate(PEGMEA), tetraethylene glycol diacrylate (TEGDA), poly(ethylene glycol)dimethacrylate (EGDMA), or N,N′-methylene-bis-acrylamide (NMBA).

Optionally, a drag compound comprises a protein or polypeptide,including BSA, lysozyme, beta-actin, myosin, lactalbumin, ovalbumin,beta-galactosidase, or lactate dehydrogenase. In some embodiments, adrag compound can be attached to a nucleic acid by an amine or thiollinkage.

In some embodiments, a mobility-altered nucleic acid comprises a nucleicacid attached to a binding partner, such as an affinity moiety forinteraction with a receptor moiety. In some embodiments, a receptormoiety can serve as a drag compound. In some embodiments, an affinitymoiety can be attached to a nucleic acid, and the affinity moiety (whichserves as the drag compound) interacts with a receptor moiety. Forexample, a nucleic acid can be attached to a biotin moiety which canbind an avidin-like moiety. An avidin-like moiety can serve as a dragcompound. An avidin-like moiety includes avidin and any derivatives,analogs and other non-native forms of avidin that can bind to biotinmoieties. Other examples of binding partners include epitopes (e.g.,protein A) and their respective antibodies (e.g., anti-FLAG antibodies),and fluorescein and anti-fluorescein antibodies. One skilled in the artwill readily recognize other binding partner combinations for attachinga drag compound to a nucleic acid.

Optionally, the drag compound can be attached to the primer by attachingthe drag compound and the primer to each of two members of a pair ofbinding partner.

Optionally, at least one primer in the reaction mixture includes biotin.

Optionally, the drag compound includes avidin or streptavidin.

Optionally, the drag compound comprises a saccharide moiety, apolysaccharide, a protein, a glycoprotein or polypeptide.

Optionally, the drag compound comprises BSA, lysozyme, beta-actin,myosin, lactalbumin, ovalbumin, beta-galactosidase, lactatedehydrogenase or immunoglobulin (e.g., IgG).

Optionally, the reaction mixture further includes an accessory protein.

Optionally, the accessory protein comprises a helicase, asingle-stranded binding protein, or a recombinase loading factor.

Optionally, the helicase comprises a uvsW from T4 phage.

Optionally, the single stranded binding protein comprises Sso SSB fromSulfolobus solfataricus, MjA SSB from Methanococcus jannaschii, or E.coli SSB protein.

Optionally, the single stranded binding protein comprises gp32 proteinfrom T4 phage or a modified gp32 protein from T4 phage.

Optionally, the recombinase loading protein comprises uvsY from T4phage.

Optionally, the reaction mixture further includes ATP.

Optionally, the reaction mixture further includes an ATP regenerationsystem.

Optionally, the ATP regeneration system includes phosphocreatine.

Optionally, the ATP regeneration system includes creatine kinase.

Optionally, the reaction mixture further includes an additive compoundfor enhancing the efficiency of, or yield, of a nucleic acidamplification reaction.

Optionally, the additive compound comprises betaine, DMSO, proline,trehalose, MMNO (4-methylmorpholine N-oxide) or a PEG-like compound.

Optionally, at least one polynucleotide template in the reaction mixtureincludes both a first sequence that is complementary or identical to atleast some portion of the first primer, and a second sequence that iscomplementary or identical to at least some portion of the secondprimer. Optionally, the reaction mixture includes a plurality of doublestranded polynucleotides containing both a first sequence that iscomplementary or identical to at least some portion of the first primer,and a second sequence that is complementary or identical to at leastsome portion of the second primer. Optionally, the first sequence islocated at or near an end of at least one double stranded polynucleotideof the plurality, and the second sequence is located at or near anotherend of at least one double stranded polynucleotide of the plurality.

Optionally, the reaction mixture further includes a diffusion-limitingagent.

Optionally, the diffusion-limiting agent reduces the rate of diffusionof the polynucleotide away from the support.

Optionally, the diffusion-limiting agent reduces the level of polyclonalnucleic acid populations attached to the support.

Optionally, the diffusion-limiting agent comprises a polymer compound.

Optionally, the diffusion-limiting agent comprises a saccharide polymer.

Optionally, the diffusion-limiting agent comprises a cellulose-basedcompound.

Optionally, the diffusion-limiting agent comprises a glucose orgalactose polymer.

Optionally, the saccharide polymer comprises cellulose, dextran, starch,glycogen, agar or agarose.

Optionally, the diffusion-limiting agent comprises a block copolymercompound.

Optionally, the diffusion-limiting agent comprises a central chain ofpoly(propylethylene oxide) flanked by two hydrophilic chains ofpoly(ethylene oxide).

Optionally, the diffusion-limiting agent forms a micelle.

Optionally, the diffusion-limiting agent forms a micellar liquidcrystal.

Optionally, the diffusion-limiting agent comprises a Pluronics'compound.

Optionally, the reaction mixture further includes a diffusion-reducingagent at a concentration of about 0.025-0.8% w/v, or about 0.05-0.7%w/v, or about 0.075-0.6% w/v, or about 0.1-0.5% w/v, or about 0.2-0.4%w/v.

Optionally, the compositions, as well as related systems, apparatuses,kits and methods, for nucleic acid amplification further include asurface, matrix or medium including a plurality of sites, at least oneof the sites being operatively coupled to one or more sensors.

Optionally, the plurality of sites comprise reaction chambers, supports,particles, microparticles, spheres, beads, filters, flowcells, wells,grooves, channel reservoirs, gels or inner wall of a tube.

Optionally, the plurality of sites can be arranged in a random ororganized array.

Optionally, the plurality of sites can be in fluid communication witheach other.

Optionally, at least one of the plurality of sites includes athree-dimensional chemical matrix.

Optionally, at least one of the plurality of sites can be covalentlyattached to a three-dimensional chemical matrix.

Optionally, at least one of the plurality of sites includes anacrylamide layer.

Optionally, at least one of the plurality of sites includes a nucleicacid that is covalently attached to an acrylamide layer.

In some embodiments, the site comprises a hydrophilic polymer matrixconformally disposed within a well operatively coupled to the sensor.

Optionally, the hydrophilic polymer matrix includes a hydrogel polymermatrix.

Optionally, the hydrophilic polymer matrix is a cured-in-place polymermatrix.

Optionally, the hydrophilic polymer matrix includes polyacrylamide,copolymers thereof, derivatives thereof, or combinations thereof.

Optionally, the polyacrylamide is conjugated with an oligonucleotideprimer.

Optionally, the well has a characteristic diameter in a range of 0.1micrometers to 2 micrometers.

Optionally, the well has a depth in a range of 0.01 micrometers to 10micrometers.

In some embodiments, the sensor includes a field effect transistor(FET). The FET can include an ion sensitive FET (ISFET),chemical-sensitive field-effect transistor (chemFET), or biologicallyactive field-effect transistor (bioFET).

Optionally, the one or more sensors are configured to detect a byproductof a nucleotide incorporation.

Optionally, the one or more sensors can be configured to detect thepresence of a chemical moiety at one or more of the plurality of sites.

Optionally, the one or more sensors comprise field-effect transistors(FET), ion-sensitive field-effect transistors (ISFET),chemical-sensitive field-effect transistors (chemFET), or biologicallyactive field-effect transistors (bioFET).

In some embodiments, the FET includes a floating gate structurecomprising a plurality of conductors electrically coupled to one anotherand separated by dielectric layers, and the floating gate conductor isan uppermost conductor in the plurality of conductors.

In some embodiments, the floating gate conductor includes an uppersurface defining a bottom surface of the site.

In some embodiments, the floating gate conductor comprises anelectrically conductive material, and the upper surface of the floatinggate conductor includes an oxide of the electrically conductivematerial.

In some embodiments, the floating gate conductor is coupled to the atleast one reaction chamber via a sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

Optionally, the byproduct from a nucleotide incorporation reactioncomprises pyrophosphate, hydrogen ions, or protons.

Also provided herein are compositions comprising any one or any subsetor all of the following: at least one reverse nucleic acid strand, aplurality of forward primers immobilized on at least one support, aplurality of reverse primers in solution, and a polymerase. The forwardand/or reverse primers are optionally low-melt or rich is adenine,thymine or uracil as described herein. An exemplary compositioncomprises a solid support comprising a plurality of spatially-separatedclonal populations each comprising a low-melt primer-binding sequence atthe 3′ end and a low-melt primer sequence on the 5′ end. Optionally, thecomposition further comprises a recombinase. Alternatively, thecomposition is optionally free of another enzyme that is not apolymerase, e.g., a recombinase or reverse transcriptase or helicase ornicking enzyme. Another exemplary composition comprises any one or moreof: (1) a reverse nucleic acid strand, (2) a plurality of low-meltforward primers immobilized on a support, (3) a plurality of low-meltreverse primers in solution, and (4) a polymerase. Optionally, theforward primers are hybridizable (e.g., complementary) to a 3′ portionor end of the reverse strand. Optionally, the reverse primers aresubstantially identical to a 5′ portion or end of the reverse strand.The composition can contain any one or more reagents described herein,and/or be subjected to any one or more procedures or conditionsdescribed herein.

In some embodiments, the disclosure relates generally to compositionscomprising amplified nucleic acids produced by any of the methods of thedisclosure. In some embodiments, a localized clonal population of clonalamplicons is formed around a discrete site on the support. An exemplarydiscrete site is a point of attachment of an initial nucleic acid strandto the support, and from which other nucleic acids within the clonalpopulation are directly or indirectly generated by primer extension,using the initial nucleic acid or its copies as a template.

Optionally the composition comprises a collection of nucleic acidsproducible by any one or more methods described herein. For example, thecollection can comprise immobilized nucleic acids which occupy one ormore distinct areas on a surface. In some embodiments, each areacomprises a plurality of identical nucleic acid strands and optionally,a plurality of identical complementary strands hybridized thereto, wherethe complementary strands have no attachment or linkage or associationwith the solid support except by virtue of hybridization to theimmobilized nucleic acid. Optionally, an individual nucleic acid strandwithin such an area is located so that another nucleic acid strand islocated on the surface within a distance of the length of that strand.Optionally there is at least one distinct area present per mm² ofsurface on which the nucleic acids are immobilized. For example thenumber of distinct areas/mm² of surface on which the nucleic acids areimmobilized is greater than 10², greater than 10³, greater than 10⁴,greater than 10⁵, greater than 10⁶, greater than 10⁷, or greater than10⁸.

The collections of amplified clonal populations can form arrays, whichcan be one-dimensional (e.g., a queue of generally monoclonalmicrobeads) or two-dimensional (e.g., the amplified clonal populationsare situated on a planar support), or three-dimensional. The individualclonal populations of an array are optionally but not necessarilysituated or arranged such that they are addressed or addressable.Optionally, different clonal populations are spaced at an appropriatedistance from one another, which distance is generally sufficient topermit different clonal populations to be distinguished from each other.In an embodiment, localized clonal populations are scattered in anordered or disordered, e.g., random, pattern over a planar substrate.

The features of an exemplary array are individual distinguishable clonalpopulations of nucleic acids, where optionally the features aredistributed over one or more supports. In an exemplary microbeadembodiment, an array comprises a plurality of microbeads, where anindividual microbead generally comprises a monoclonal population ofnucleic acids, and different microbeads generally comprise differentclonal populations (e.g., which differ in sequence). Optionally, themicrobeads are distributed or packed in a monolayer over a planarsubstrate. In other embodiments, the array comprises a single (e.g.,planar) support, the single support comprising a plurality of spatiallydiscrete clonal populations of nucleic acids, where different clonalpopulations optionally differ in sequence.

Optionally, one or more nucleic acids within individual clonalpopulations can be attached to the planar substrate directly. In anotherexample, the nucleic acids of individual clonal populations are attachedto microbeads, for example as discussed herein. The clonal microbeadsare optionally packed closely together over a planar substrate, inrandom or ordered fashion. Optionally, more than 20%, 30%, 50%, 70%,80%, 90%, 95% or 99% of the microbeads are in contact with at least one,two, four or six other microbeads. Optionally, less than 10%, 20%, 30%,50%, 70%, 80%, 90%, 95% or 99% of the microbeads are in contact withone, two, four or six other microbeads.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, for nucleicacid amplification, comprising conducting a multiplex nucleic acidamplification using any of the amplification methods, compositions orsystems disclosed herein.

In some embodiments, the methods include performing multiplexamplification using a recombinase (e.g., a recombinase-mediatedmultiplex nucleic acid amplification reaction).

In some embodiments, the methods can further include re-amplifying theamplicons from the multiplex nucleic acid amplification using a nucleicacid amplification reaction,

Optionally, a multiplex nucleic acid amplification can be conducted in asingle reaction mixture.

Optionally, a multiplex nucleic acid amplification can be conducted on asample containing a plurality of different nucleic acid targetsequences.

Optionally, a plurality of different nucleic acid target sequences canbe amplified in a single reaction mixture.

Optionally, at least dozens, or at least hundreds, or at least thousands(or more) of nucleic acid target sequences can be amplified in thesingle reaction mixture.

Optionally, at least fifty, or at least one hundred nucleic acid targetsequences can be amplified in the single reaction mixture.

Optionally, the multiplex amplifying can include contacting at least aportion of the sample with any one or any combination of a recombinase,a polymerase and/or at least one primer.

Optionally, the multiplex amplifying can be conducted under isothermalor thermocycling conditions.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, for nucleicacid amplification, comprising multiplex nucleic acid amplification,which includes amplifying within a single reaction mixture at leastfifty different nucleic acid target sequences (or more) from a samplecontaining a plurality of different nucleic acid target sequences, theamplifying including contacting at least a portion of the sample with arecombinase and a plurality of primers under isothermal amplificationconditions.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, for nucleicacid amplification, comprising multiplex nucleic acid amplification,which includes amplifying within a single reaction mixture differentnucleic acid target sequences from a sample containing a plurality ofdifferent nucleic acid target sequences, the amplifying includinggenerating a plurality of at least fifty different amplified targetsequences (or more) by contacting at least a portion of the sample witha polymerase and a plurality of primers under isothermal amplificationconditions.

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, for nucleicacid amplification, comprising generating substantially monoclonalnucleic acid populations by re-amplifying the amplicons from themultiplex nucleic acid amplification using a nucleic acid amplificationreaction (e.g., a recombinase).

Optionally, methods for multiplex nucleic acid amplification can furtherinclude a recombinase-mediated nucleic acid amplification method whichincludes re-amplifying at least some of the at least fifty differentamplified target sequences by: (a) forming a reaction mixture includinga single continuous liquid phase containing (i) a plurality of supports,(ii) at least one of the fifty different amplified target sequences and(iii) a recombinase; and (b) subjecting the reaction mixture toamplification conditions, thereby generating a plurality of supportsattached to substantially monoclonal nucleic acid populations attachedthereto.

Optionally, in methods for multiplex nucleic acid amplification, thedifferent nucleic acid target sequences from the sample can be amplifiedunder conditions that are substantially non-exhaustive.

Optionally, in methods for multiplex nucleic acid amplification, thedifferent nucleic acid target sequences from the sample can be amplifiedunder conditions that are substantially exhaustive.

Optionally, in methods for multiplex nucleic acid amplification, thesingle reaction mixture comprises an isothermal or a thermocyclingreaction mixture.

In some embodiments, two or more of the template or targets can beamplified within separate chambers, wells, cavities or sites of an arraythat are in fluid communication with each other, or that are occupied bythe same single continuous liquid phase of an amplification reactionmixture. Such embodiments include embodiments for array-based nucleicacid amplification.

For example, in some embodiments the disclosure relates to methods fornucleic acid amplification, comprising: distributing a targetpolynucleotide into a reaction chamber or site in an array of reactionchambers or sites, and amplifying the single target polynucleotidewithin the reaction chamber or site. Optionally, two or more targetpolynucleotides are distributed into two or more reaction chambers orsites of the array, and two or more of the distributed targetpolynucleotides are amplified in parallel within their respectivereaction chambers or sites. Optionally, at least two of the reactionchambers or sites each receive a single target polynucleotide during thedistributing (one or more of the reaction chambers or sites canoptionally receive zero or more than one target polynucleotide duringthe distributing). At least two target polynucleotides can be clonallyamplified within their respective reaction chambers. At least one of thereaction chambers including a target polynucleotide can be in fluidcommunication with at least one other reaction chamber including atarget polynucleotide during the amplifying.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems, apparatuses and kits) for nucleicacid amplification, comprising: (a) distributing at least two differentpolynucleotides into an array of reaction chambers by introducing asingle one of said polynucleotides into at least two of said reactionchambers that are in fluid communication with each other; and (b)forming at least two substantially monoclonal nucleic acid populationsby amplifying the polynucleotides within said at least two reactionchambers. Typically, the at least two reaction chambers remain in fluidcommunication with each other during the amplifying.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems, apparatuses and kits) for nucleicacid amplification, comprising (a) in an array of reaction chambersincluding a first and a second reaction chamber, distributing a firsttemplate polynucleotide into the first reaction chamber and a secondtemplate polynucleotide into the second reaction chamber, and (b)forming at least two substantially monoclonal nucleic acid population byclonally amplifying the first and second template polynucleotides withintheir respective reactions chamber, where the single polynucleotide isdistributed from a nucleic acid sample having multiple differentpolynucleotides. Optionally, the first and second reaction chambersinclude different portions of a single continuous liquid phase duringthe amplifying. For example, the first and second reaction chambers ofthe array can be in fluid communication during the amplifying.

In some embodiments, the disclosure relates generally to methods fornucleic acid amplification, comprising (a) distributing a differentsingle polynucleotide into each of a plurality of reaction chambers, and(b) forming a monoclonal nucleic acid population in each of the reactionchambers by amplifying the different single polynucleotides within theplurality of reaction chambers, where the single differentpolynucleotides are distributed from a nucleic acid sample havingmultiple different polynucleotides.

In some embodiments, the disclosure relates generally to methods fornucleic acid amplification, comprising (a) distributing at least twodifferent polynucleotides into an array of reaction chambers byintroducing a single one of said polynucleotides into at least two ofsaid reaction chambers that are in fluid communication with each other;and (b) forming at least two substantially monoclonal nucleic acidpopulations by amplifying the polynucleotides within said at least tworeaction chambers.

In some embodiments, the methods can further include introducing one ormore supports (e.g., beads or particles and the like) into at least onereaction chamber or site of the array. The one or more supports can beintroduced into the at least one reaction chamber or site prior to,during or after the polynucleotides are distributed into the array. Insome embodiments, at least one reaction chamber or site of the arrayreceives a single support. In some embodiments, the majority of reactionchambers or sites receive a single support. In some embodiments, thesupports can be mixed with the polynucleotides prior to thedistributing, and distributed into the array together with thepolynucleotides. At least one support can optionally be linked to anucleic acid molecule comprising a primer sequence that is substantiallycomplementary or substantially identical to a portion of apolynucleotide present in the reaction chamber or site during theamplifying. In some embodiments, the at least one support includes anucleic acid molecule comprising a primer sequence that is substantiallycomplementary or substantially identical to a portion of a targetpolynucleotide or template in the reaction chamber or well. In someembodiments, the at least one support includes a nucleic acid moleculecomprising a primer sequence that is substantially complementary orsubstantially identical to at least a portion of another primer presentin the reaction chamber or site during the amplifying.

In some embodiments, the amplifying can include introducing a reactionmixture into at least one reaction chamber or site in the array. Thereaction mixture is optionally introduced into the reaction chamber orsite prior to, during or after the distributing of the polynucleotidesinto the array, or the introducing of the supports into the array. Thereaction mixture, supports and polynucleotides can be introduced ordistributed into the array in any order or any combination. In someembodiments, at least one reaction chamber or site of the array receivesa single support, a single polynucleotide and sufficient reactionmixture to support amplification of the polynucleotide within thereaction chamber or site.

In some embodiments, the method can include hybridizing at least aportion of the polynucleotide to the support by contacting the supportwith the polynucleotide under hybridization conditions. The hybridizingcan occur before, during or after introduction of the supports and/orpolynucleotides into the reaction chambers or sites of the array. Insome embodiments, at least one support linked to a first primingsequence is introduced into at least one reaction chamber or site of thearray, and then a polynucleotide is introduced into the reaction chamberor site, and the polynucleotide is hybridized to the support within thereaction chamber or site. Alternatively, the support can hybridize to anamplification product generated via amplification of the polynucleotidewithin the reaction chamber or site.

The reaction mixture can include any of the reaction mixtures andcomponents described herein. In some embodiments, the reaction mixtureincludes any one or more of the following components: Isothermalamplification reagents (e.g., one or more recombinases, helicases andrelated accessory factors, polymerases and the like), sieving agents,nucleotides, and the like.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, kits, systems and apparatuses) for nucleic acidamplification, comprising: (a) introducing, in any order or combination,a first polynucleotide template and a first support into a firstreaction chamber or site of an array of reaction chambers or sites, anda second polynucleotide template and a second support into a secondreaction chamber or site of the array; and (b) clonally amplifying thefirst polynucleotide template onto the first support within the firstreaction chamber or site, and the second polynucleotide template ontothe second support within the second reaction chamber or site, while thefirst reaction chamber or site is in fluid communication with the secondreaction chamber or site during the amplifying. The clonally amplifyingcan include generating a first support attached to a first ampliconderived from the first polynucleotide template, and a second supportattached to a second amplicon derived from the second polynucleotidetemplate. Optionally, both the first and second sites (or reactionchambers) include the same continuous liquid phase of the same reactionmixture during the amplifying. For example, the reaction mixture caninclude a single continuous liquid phase that includes both the firstand second polynucleotide templates and the first and second supports.The reaction mixture can be introduced into the reaction chambers orsites of the array before, during or after introduction of thepolynucleotide template and/or the support. In some embodiments, thedisclosed method further includes introducing the reaction mixture intothe first and second reaction chambers or sites after introducing thefirst and second polynucleotides templates and the first and secondsupports. In some embodiments, the reaction mixture includes arecombinase or a helicase or both a recombinase and helicase. Therecombinase can be derived from a myoviral (e.g., uvsX), bacterial,yeast or human recombinase, or analog thereof from another species. Insome embodiments, the reaction mixture includes a polymerase. In someembodiments, the reaction mixture includes a sieving agent, for examplepolyacrylamide, agarose or a cellulose polymer (e.g., HEC, CMC or MC, orderivatives thereof). In some embodiments, the reaction mixture includesa diffusion-limiting agent.

In some embodiments, the amplifying includes linking a polynucleotidetemplate to a support or surface (e.g., a particle or bead) having afirst primer containing a first primer sequence, by hybridizing thefirst primer binding sequence of said polynucleotide template to a firstprimer sequence in the first primer of the support.

In some embodiments, methods for nucleic acid amplification can beconducted in a single continuous liquid phase that does not providecompartmentalization of the multiple nucleic acid amplificationreactions occurring in a single reaction vessel. In some embodiments,methods for nucleic acid amplification can be conducted in water-in-oilemulsions that provide compartmentalization (micro-reactors).

In embodiments where the amplifying is performed within the reactionchambers or sites of an array, as well as embodiments where theamplifying is performed within a single reaction vessel, the surface orsupport optionally includes at least a first primer including a firstprimer sequence. In some embodiments, one or more of the polynucleotidetemplates include a first primer binding sequence. The first primerbinding sequence can be identical, or substantially identical, to thefirst primer sequence. Alternatively, the first primer binding sequencecan be complementary, or substantially complementary, to the firstprimer sequence. In some embodiments, the first primer sequence and thefirst primer binding sequence do not exhibit significant identity orcomplementarity, but instead are substantially identical to, orsubstantially complementary to, another nucleotide sequence present inthe reaction mixture. In such embodiments, amplification can includeformation of an amplification reaction intermediate that includesnucleotide sequences having significant identity, or complementarity to,the first primer sequence, the first primer binding sequence, or both.

In some embodiments, at least two different polynucleotide templates arepresent in the reaction mixture, and the amplification results in theformation of at least two different substantially monoclonalpopulations, each derived from amplification of a single one of saidpolynucleotide templates. In some embodiments, two or more of the atleast two substantially monoclonal populations are attached to the samesupport or surface. Each of the two or more substantially monoclonalpopulations can be attached to a different unique location on the samesupport or surface. Alternatively, each of the two or more substantiallymonoclonal populations can each be attached to a different support orsurface. Segregation of different monoclonal populations to differentsupports or surfaces can be advantageous in applications requiringsegregation of the populations prior to analysis. In some embodiments,the support or surface is part of a bead or particle, which can bespheroid or spherical in shape. In some embodiments, the support orsurface forms part of a two-dimensional or three-dimensional array.

In some embodiments, the disclosed methods for nucleic acidamplification can be performed while including a sieving agent or adiffusion-reducing agent within the reaction mixture. These agents canincrease the total number and/or proportion (e.g., percentage) ofmonoclonal populations formed during the amplification. In someembodiments, the methods include use of reaction mixtures providing forincreased yields of monoclonal or substantially monoclonal ampliconsrelative to conventional reaction mixtures.

In some embodiments, the methods include amplifying by partiallydenaturing the templates. For example, the amplifying can includetemplate walking. For example, the templates to be amplified can includean adapter sequence containing a primer binding site that has arelatively low Tm compared to the template as a whole. In someembodiments, amplification is performed at temperatures that aresubstantially higher than the Tm of adapter sequence but aresubstantially lower than the Tm of the template, as described in moredetail herein. In some embodiments, amplification is performed attemperatures that are at least 5° C., 10° C., 15° C., 20° C., 25° C. or50° C. below the Tm of the nucleic acid template. In some embodiments,amplification is performed at temperatures greater (for example, atleast 5° C., 10° C., 15° C., 20° C., 25° C. or 50° C. greater) than theTm of the first primer, the second primer, or both the first and secondprimer.

In some embodiments, the reaction mixtures can optionally include anyone or more of the following: (a) one or more supports, optionallyincluding at least a first primer sequence; (2) a recombinase; (3) apolymerase; (4) a diffusion limiting agent; (5) a sieving agent; (6) acrowding agent; (7) an ATP regeneration system; (8) a single strandedbinding protein (SSBP); and (8) a recombinase accessory factor, forexample a recombinase loading protein. In some embodiments, the yield ofmonoclonal, or substantially monoclonal, populations is increased byamplifying the polynucleotide templates onto a surface (for example, byattaching one of the amplification primers to the surface) in thepresence of a diffusion-limiting and/or a sieving agent. The diffusionlimiting agent, or the sieving agent, can provide for increased yieldsof monoclonal populations by reducing diffusion or migration ofamplified product polynucleotides away from the surface during theamplification.

In some embodiments, the reaction mixture includes one or moreisothermal amplification reagents. Such reagents can include, forexample, recombinases or helicases.

In some embodiments, methods for nucleic acid amplification can beconducted with an enzyme that catalyzes homologous recombination, forexample an enzyme that can bind a first primer to form a complex or cancatalyze strand invasion or can form a D-loop structure. In someembodiments, an enzyme that catalyzes homologous recombination comprisesa recombinase.

In some embodiments, amplification conditions include isothermalconditions or thermocycling conditions.

In some embodiments, methods for nucleic acid amplification comprise:(a) forming a reaction mixture including a single continuous liquidphase containing (i) an enzyme that catalyzes homologous recombination,(ii) one or a plurality of surfaces, and (iii) a plurality of differentpolynucleotides; and (b) subjecting the reaction mixture to a conditionsuitable for nucleic acid amplification.

In some embodiments, methods for nucleic acid amplification comprise:(a) forming a reaction mixture including a single continuous liquidphase containing (i) an enzyme that catalyzes homologous recombination,(ii) one or a plurality of beads each attached to a plurality of a firstprimers, and (iii) a plurality of different polynucleotides; (b) formingtwo or more substantially monoclonal amplified nucleic acid populationsby subjecting the reaction mixture to amplification conditions. Theamplification conditions can include isothermal or thermocylingconditions. In some embodiments, the first primers can hybridize to atleast a portion of the polynucleotides.

In some embodiments, the disclosure relates generally to a method fornucleic acid amplification, comprising: (a) forming a reaction mixtureincluding a single continuous liquid phase containing one or moresupports (or surfaces), a plurality of polynucleotides and arecombinase; (b) clonally amplifying at least two of said plurality ofdifferent polynucleotides onto at least one support (or surface) bysubjecting the reaction mixture to amplification conditions. In someembodiments, the amplification conditions can include isothermal orthermocyclic amplification conditions. The reaction mixture canoptionally include a recombinase. In some embodiments, the reactionmixture includes a polymerase. In some embodiments, the reaction mixtureincludes a primer, which can be in solution. Optionally, at least one ofthe supports or surfaces can include a primer.

In some embodiments, the disclosure relates generally to a method fornucleic acid amplification comprising: (a) forming a reaction mixtureincluding a single continuous liquid phase containing (i) a recombinase,(ii) a plurality of beads attached or one or more first primersincluding a first primer sequence, and (iii) a plurality of differentpolynucleotide templates; (b) hybridizing at least one of said firstprimers to at least one of the plurality of different polynucleotidetemplates; (c) subjecting the reaction mixture to a nucleic acidamplification conditions and generating at least one substantiallymonoclonal polynucleotide population by amplifying at least one of thepolynucleotide templates to form at least a first amplified population.In some embodiments, at least 30%, 90% of the polynucleotides in the atleast one substantially monoclonal population are substantiallyidentical (or substantially complementary) to at least one of thepolynucleotide templates originally present in the reaction mixture. Insome embodiments, at least a portion of the first amplified populationis attached to one bead of the plurality of beads.

In some embodiments, forming a reaction mixture in step (a) comprises:forming a nucleoprotein complex by contacting the recombinase with atleast one of the plurality of the first primers which are attached tothe plurality of beads.

In some embodiments, the subjecting the reaction mixture to a nucleicacid amplification conditions in step (b) comprises conducting anucleotide polymerization reaction. For example, a nucleotidepolymerization reaction can include incorporating a nucleotide into afirst primer sequence, optionally when the first primer sequence ishybridized to one of the polynucleotide templates in the reactionmixture.

In some embodiments, subjecting the reaction mixture to a nucleic acidamplification conditions includes contacting the first primer with apolynucleotide template, a recombinase, a polymerase and nucleotides, inany order or in any combinations.

In some embodiments, the nucleic acid amplification conditions includerepeating cycles of: forming a nucleoprotein complex including arecombinase, at least a portion of the a first primer, and at least aportion of a first polynucleotide template, and contacting thenucleoprotein complex with a polymerase that catalyzes the incorporationof one or more nucleotides into the first primer.

In some embodiments, recurring nucleic acid amplification reactions canbe conducted to generate a plurality of beads each attached with asubstantially monoclonal population of polynucleotides.

In some embodiments, methods for nucleic acid amplification can beconducted under isothermal conditions or thermocycling conditions.

In some embodiments, the plurality of different polynucleotides can besingle- or double-stranded polynucleotides. In some embodiments, heat orchemical denaturation of double-stranded polynucleotides is notnecessary because the recombinase can generate localized stranddenaturation by catalyzing strand invasion.

In some embodiments, methods for nucleic acid amplification can beconducted in a single reaction vessel. In some embodiments, a nucleicacid amplification reaction can be conducted in a single reaction vesselcomprising a single continuous liquid phase. For example, the singlecontinuous liquid phase can include an amplification mixture comprisinga plurality of beads each attached with a plurality of a first primer, aplurality of different polynucleotides, and a plurality of recombinases.In some embodiments, an amplification mixture can further include apolymerase and a plurality of nucleotides. In some embodiments, anamplification mixture can further include ATP, nucleotides andco-factors. Non-limiting examples of a single reaction vessel include atube, well or similar structures.

In some embodiments, polynucleotides and reagents can be deposited intoa reaction vessel in any order, including sequentially or substantiallysimultaneously or a combination of both. In some embodiments, reagentsinclude a beads attached with multiple first primers, recombinases,polymerases, nucleotides, ATP, divalent cations, and co-factors.

In some embodiments, methods for nucleic acid amplification can beconducted in a single continuous liquid phase. The single continuousliquid phase can include any liquid phase wherein any given portion orregion of the single liquid continuous phase is in fluid communicationwith any other portion or region of the same single liquid continuousphase. Typically, components that are dissolved or suspended in thesingle continuous liquid phase can freely diffuse or migrate to anyother point in the liquid phase. In some embodiments, however, thesingle continuous liquid phase can include diffusion limiting agentsthat slow down the rate of diffusion within the single continuous liquidphase. One exemplary embodiment of a single continuous liquid phase is asingle aqueous droplet in a water-in-oil emulsion; in such an emulsion,each droplet will form a separate phase; two droplets may coalesce toform a single phase.

In some embodiments, a single continuous liquid phase consistsessentially of a single aqueous phase. In some embodiments, the singlecontinuous liquid phase lacks a non-aqueous phase; for example, thecontinuous liquid phase does not include oil or organic solvents. Insome embodiments, multiple nucleic acid amplification reactions occur inan aqueous phase in a single reaction vessel. In some embodiments, asingle continuous liquid phase does not compartmentalize the multiplenucleic acid amplification reactions occurring in a single reactionvessel.

In some embodiments, methods for nucleic acid amplification can beconducted in water-in-oil emulsions that provide compartmentalization.

When conducting a nucleic acid amplification using a plurality ofpolynucleotide templates, clonal amplification using traditionalamplification methods typically relies on techniques such ascompartmentalization of the reaction mixture into segregated portions orcomponents that are not in fluid communication with each other in orderto maintain clonality and prevent cross-contamination of differentamplified populations and to maintain adequate yields of monoclonalamplified product. Using such conventional amplification methods, it istypically not feasible to clonally amplify polynucleotide templateswithin the same reaction mixture without resorting tocompartmentalization or distribution of the reaction mixture intoseparate compartments or vessels, because any polynucleotides within thereaction mixture (including templates and/or amplified products) willtend to migrate randomly through the mixture due to diffusion and/orBrownian motion during such amplification. Such diffusion or migrationtypically increases the incidence of polyclonal amplification and thusvery few, if any, monoclonal populations will be produced.

One suitable technique to reduce the production of polyclonalpopulations in conventional amplification methods uses physical barriersto separate individual amplification reactions into discretecompartments. For example, emulsion PCR uses water-in-oil microreactors,where an oil phase includes many separate, i.e., discontinuous, aqueousreaction compartments. Each compartment serves as an independentamplification reactor, thus the entire emulsion is capable of supportingmany separate amplification reactions in separate (discontinuous) liquidphases in a single reaction vessel (e.g., an Eppendorf tube or a well).Similarly, an amplification “master mix” can be prepared and distributedinto separate reaction chambers (e.g., an array of wells), creating aset of discrete and separate phases, each of which defines a separateamplification reaction. Such separate phases can be further sealed offfrom each other prior to amplification. Such sealing can be useful inpreventing cross-contamination between parallel and separate reactions.Exemplary forms of sealing can include use of lids or phase barriers(e.g., mineral oil layer on top of an aqueous reaction) tocompartmentalize the PCR reactions into individual and discretecompartments, between which transfer of reaction components does notoccur.

In some embodiments, the nucleic acid amplification reaction can beconducted in an emulsion that can effectively increase the diffusiondistance between nucleic acid template molecules. In some embodiments,the emulsion includes a mixture of an aqueous liquid and awater-immiscible organic liquid. Optionally, the emulsion comprises atleast one anionic, cationic or non-ionic surfactant. In someembodiments, the emulsion can be a microemulsion (Danielsson and Lindman1981 Colloids Surf. A 3:391; U.S. Pat. No. 5,151,217). In someembodiments, the microemulsion can have a droplet-type dispersioncomprising oil-in-water, water-in-oil, or a bicontinuous microemulsion.In some embodiments, a bicontinuous microemulsion comprises a waterimmiscible organic liquid forming a first continuous phase. In someembodiments, a bicontinuous microemulsion comprises an aqueous liquidforming a second continuous phase. In some embodiments, the first andthe second continuous phases are entangled together to form asponge-like dispersion of water and oil.

In some embodiments, the water immiscible organic liquid comprises anoil. In some embodiments, the oil can be from a natural source,including animal (e.g., tallow or lard), fish (e.g., fish oil), shark,seeds, nuts or plants (e.g., vegetable oils). In some embodiments, theoil can be from derived from petroleum, including mineral oils. In someembodiments, the oil comprises a fluorochemical oil, polyalphaolefin orester oil.

In some embodiments, the surfactant includes small molecule surfactants,polymeric surfactants, triblock co-polymer surfactants or non-ionicblock copolymer surfactants. Optionally, the surfactant comprises asorbitan oleate or a silicone surfactant.

In some embodiments, the bicontinuous microemulsions can be formulatedwith a high % (w/w) of at least one surfactant to create the sponge-likedispersion having a continuous aqueous phase entangled with a continuouswater immiscible organic phase. Optionally, a bicontinuous microemulsioncan be formulated with about 1% (w/w) to about 20% (w/w) surfactant, orabout 10% (w/w) to about 20% (w/w) surfactant, or about 15% (w/w) toabout 20% (w/w) surfactant. Optionally, the bicontinuous microemulsioncan be formulated with at least one co-surfactant.

Optionally, the formulation for a bicontinuous microemulsion includes2,2,4-trimethylpentane (TMP) and polyoxyethylene lauryl ether (e.g.,Brij) (U.S. Pat. No. 6,429,200).

Optionally, the formulation for a bicontinuous microemulsion includes apolyalphaolefin, a polymeric surfactant, and a small moleculesurfactant. Optionally, the combined % w/w of the polymeric and smallmolecule surfactants can be about 10% (w/w) to about 20% (w/w).

Optionally, the formulation for a bicontinuous microemulsion includes amineral oil, an ester oil and a silicone surfactant. Optionally, the %w/w of the silicone surfactant can be about 10% (w/w) to about 20%(w/w).

Optionally, the bicontinuous microemulsions include toluene, Triton-X100and water, for example including at least 6% (w/w) Triton X-100 (Liu2003 Langmuir 19:7249-7258).

Other techniques to prevent cross-contamination and reduce polyclonalityrely on immobilization of one or more reaction components (for example,one or more templates and/or primers) during amplification to preventcross contamination of amplification reaction products and consequentreduction in monoclonality. One such example includes bridge PCR, whereall of the primers required for amplification (e.g., forward and reverseprimer) are attached to the surface of a matrix support. In addition tosuch immobilization, additional immobilization components can beincluded in the reaction mixture. For example, the polynucleotidetemplate and/or amplification primers cam be suspended in gels or othermatrices during the amplification so as to prevent migration ofamplification reaction products from the site of synthesis. Such gelsand matrices typically require to be removed subsequently, requiring theuse of appropriate “melting” or other recovery steps and consequent lossof yield.

In some embodiments, the disclosure provides methods for performingsubstantially clonal amplification of multiple polynucleotide templatesin parallel in a single continuous liquid phase of a reaction mixture,without need for compartmentalization or immobilization of multiplereaction components (e.g., both primers) during amplification. Instead,mixtures of polynucleotide templates in solution can be directlycontacted with amplification reaction components and a suitable surfaceor support having a first primer attached thereto, Other componentsrequired for amplification can be provided in the same continuous liquidphase, including a polymerase, one or more types of nucleotide andoptionally a second primer. In some embodiments, the reaction mixturealso includes a recombinase. Optionally, the reaction mixture furtherincludes at least one agent selected from the group consisting of: adiffusion limiting agent, a sieving agent, and a crowding agent.Examples of amplification mixtures suitable for achieving monoclonalamplification of templates contained in a single continuous liquid phaseare described further herein. Optionally, different templates can beamplified onto different locations on a single surface or support, ordifferent templates can be amplified onto different surfaces ordifferent supports within the same reaction mixture.

In some embodiments, the reaction mixture can include one or moresieving agents. The sieving agent optionally includes any compound thatcan provide a physical barrier to migration of polynucleotide templatesor their corresponding amplified products. (Migration can include anymovement of template or amplified product within the reaction mixture;diffusion includes forms of migration involving movement along aconcentration gradient). In some embodiments, a sieving agent comprisesany compound that can provide a matrix having a plurality of pores thatare small enough to reduce the movement of any one or more specificcomponents of a nucleic acid synthesis reaction mixture, or a nucleicacid reaction mixture.

In some embodiments, a sieving agent provides a molecular sieve. Forexample, a sieving agent can reduce the movement of a polynucleotide (ora polynucleotide associated with a surface or bead) through a reactionmixture containing the sieving agent. The sieving agent can optionallyhave pores.

Inclusion of a sieving agent may be advantageous when clonallyamplifying two or more template polynucleotides within a singlecontinuous liquid phase of a reaction mixture. For example, the sievingagent can prevent or slow diffusion of templates, or amplifiedpolynucleotides produced via replication of at least some portion of atemplate, within the reaction mixture, thus preventing the formation ofpolyclonal contaminants without requiring compartmentalization of thereaction mixture by physical means or encapsulation means (e.g.,emulsions) during the amplification. Such methods of clonally amplifyingtemplates within a single continuous liquid phase of a single reactionmixture without need for compartmentalization greatly reduces the cost,time and effort associated with generation of libraries amenable forhigh-throughput methods such as digital PCR, next generation sequencing,and the like.

In some embodiments, the average pore size of the sieving agent is suchthat movement of a target component within the reaction mixture (e.g., apolynucleotide) is selectively retarded or prevented. In one example,the sieving agent comprises any compound that can provide a matrixhaving a plurality of pores that are small enough to slow or retard themovement of a polynucleotide through a reaction mixture containing thesieving agent. Thus, a sieving agent can reduce Brownian motion of apolynucleotide.

In some embodiments, the sieving agent acts selectively to retardmigration of molecules having an average molecular size or weight abovea particular threshold value or range, without retarding the migrationof other molecules having an average molecular size or weight below thethreshold value or range.

In some embodiments, the sieving agent acts selectively to retardmigration of molecules having an average molecular size or weight belowa particular threshold value or range, without retarding the migrationof other molecules having an average molecular size or weight above thethreshold value or range.

In some embodiments, a sieving agent can be selected to selectivelyretard, slow down, reduce or prevent movement of a polynucleotidethrough a reaction mixture, but large enough to permit movement ofsmaller components (for example, cations, nucleotides, ATP andco-factors) through the reaction mixture. In some embodiments, thesieving agent has an average pore size or average pore size range thatcan be modulated by increasing or decreasing the concentration of thesieving agent. For example, the molecular weight, intrinsic viscosityand concentration of a sieving agent (or a combination of sievingagents) can be selected to prepare a nucleic acid reaction mixture in aparticular solvent (e.g., water) to produce a matrix having a desiredcapacity to prevent migration of target polynucleotides of a particularsize or length; or a desired average pore size or viscosity. In someembodiments, a sieving agent can reduce bulk flow by increasing theviscosity of a nucleic acid reaction mixture. In some embodiments, asieving agent can be water soluble. In some embodiments, a matrix havinga plurality of pores can be prepared by mixing a sieving agent with asolvent (e.g., an aqueous solvent, such as water). In some embodiments,a sieving agent does not interfere with formation of a recombinasenucleoprotein complex or with nucleotide polymerization.

In some embodiments, the disclosure relates generally to methods forconducting a nucleic acid amplification reaction, comprising generatingtwo or more substantially monoclonal populations by amplifying a targetpolynucleotide onto a surface or support in the presence of one or moresieving agents, optionally in the presence of a recombinase, apolymerase, or any other suitable agent capable of catalyzing orpromoting nucleic acid amplification.

In some embodiments, inclusion of a sieving agent in a reaction mixturecan reduce the movement of a polynucleotide away from a given support orsurface (e.g., reduce polynucleotide shedding) and can increase thelikelihood that the polynucleotide will hybridize to the support orsurface and provide an initiation site for nucleotide polymerization,thereby increasing the proportion of substantially monoclonal ampliconsgenerated during the amplification reaction.

In some embodiments, the amplification includes amplifying a pluralityof different polynucleotide templates onto a plurality of different beadsupports in the presence of a sieving agent, and recovering a percentageof substantially monoclonal bead supports, each such substantiallymonoclonal bead support include a bead support attached to asubstantially monoclonal polynucleotide population. In some embodiments,the percentage of substantially monoclonal bead supports recovered issubstantially greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 89%, 90%, or 95% of total amplified beadsupports (i.e., total bead supports including either polyclonal ormonoclonal populations) recovered from the reaction mixture. In someembodiments, the percentage of substantially monoclonal bead supportsrecovered is substantially greater than the percentage of substantiallymonoclonal bead supports recovered following amplification in theabsence of the sieving agent but under otherwise essentially similar orsame amplification conditions.

In some embodiments, a sieving agent comprises a polymer compound. Insome embodiments, a sieving agent comprises a cross-linked or anon-cross linked polymer compounds. By way of non-limiting examples, thesieving agent can include polysaccharides, polypeptides, organicpolymers, etc.

In some embodiments, a sieving agent comprises linear or branchedpolymers. In some embodiments, a sieving agent comprises charged orneutral polymers.

In some embodiments, the sieving agent can include a blend of one ormore polymers, each having an average molecular weight and viscosity.

In some embodiments, the sieving agent comprises a polymer having anaverage molecular weight of about 10,000-2,000,000, or about12,000-95,000, or about 13,000-95,000.

In some embodiments, a sieving agent can exhibit an average viscosityrange of about 5 centipoise to about 15,000 centipoise when dissolved inwater at 2 weight percent measured at about 25° C., or about 10centipoise to about 10,000 centipoise as a 2% aqueous solutions measuredat about 25° C., or about 15 centipoise to about 5,000 centipoise as a2% aqueous solution measured at about 25° C.

In some embodiments, a sieving agent comprises a viscosity averagemolecular weight (M_(v)) of about 25 to about 1,5000 kM_(v), or about75-1,000 kM_(v), or about 85-800 kM_(v). In some embodiments, thereaction mixture comprises a sieving agent at about 0.1 to about 20%weight per volume, or about 1-10% w/v, or about 2-5% w/v.

In some embodiments, a sieving agent comprises an acrylamide polymer,for example polyacrylamide.

In some embodiments, a sieving agent comprises a polymer of one or aminoacids. For example, the sieving agent can include polylysine,poly-glutamic acid, actin, myosin, keratin, tropmyosin, and the like. Insome embodiments, the sieving agent can include a derivative of any ofthese polypeptides.

In some embodiments, a sieving agent comprises a polysaccharide polymer.In some embodiments, a sieving agent comprises a polymer of glucose orgalactose. In some embodiments, a sieving agent comprises one or morepolymers selected from the group consisting of: cellulose, dextran,starch, glycogen, agar, chitin, pectin or agarose. In some embodiments,the sieving agent comprises a glucopyranose polymer.

In some embodiments, the sieving agent comprises a polymer having one ormore groups that are polar or charged under amplification reactionconditions. For example, the polymer can include one or more cationicgroups, one or more anionic groups, or both. In some embodiments, thesieving agent is a polysaccharide including one or more charged groups.In some embodiments, the sieving agent is a polysaccharide including oneor more carboxy groups that are, or tend to be, negatively charged underamplification reaction conditions. For example, the sieving agent caninclude carboxy-methyl cellulose (CMC) polymers. In some embodiments,the sieving agent can include spermine and/or spermidine. In someembodiments, the sieving agent includes polylysine and/or polyarginine.For example, the sieving agent can include poly-L-lysine, poly-D-lysine,poly-D-glutamic acid, and the like. In some embodiments, the sievingagent includes one or more histone proteins or histone-nucleic acidcomplexes or derivatives thereof. The histones are highly alkalineproteins that are capable of binding to nucleic acid and include theproteins H1, H2A, H2B, H3 and H4. In some embodiments, the histoneprotein is modified, for example via methylation, acetylation,phosphorylation, ubiquitination, SUMOylation, citrullination,ribosylation (including ADP-ribosylation), and the like.

In some embodiments, the sieving agent includes a polymer that includeschemically substituted polymers. The polymers can include reactivegroups (for example, which can be reacted with suitable substituents toproduce substituted polymers. In some embodiments, the polymer includesa fluoro-, carboxy-, amino-, or alkoxy-substituted polymer. In someembodiments, the polymer is modified via methylation, acetylation,phosphorylation, ubiquitination, carboxylation, and the like. Thesubstituent can include a charged group, for example an anionic orcationic group, and substitution of this group into the polymer chaincan result in generation of a charged polymer. The degree ofsubstitution can vary from between about 0.2 and about 1.0 derivativesper monomer unit, typically between about 0.4 and about 1.0, even moretypically between about 0.6 and 0.95.

In some embodiments, the sieving agent includes a cellulose derivative,such as sodium carboxymethyl cellulose, sodium carboxymethyl2-hydroxyethyl cellulose, methyl cellulose, hydroxyl ethyl cellulose,2-hydroxypropyl cellulose, carboxy methyl cellulose, hydroxyl propylcellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose,(hydroxypropyl)methyl cellulose or hydroxyethyl ethyl cellulose, or amixture including any one or more of such polymers.

In some embodiments, a nucleic acid reaction mixture comprises a mixtureof different sieving agents, for example, a mixture of differentcellulose derivatives, starch, polyacrylamide, and the like.

In some embodiments, the sieving agent includes one or more compositepolymers comprised of subportions of any two different polymers,including any of the polymers described herein. For example, thecomposite polymer can include polysaccharide polymer linked to apolynucleotide polymer, such as a polysaccharide linked to DNA or RNA.In some embodiments, the sieving agent can include a polymer comprisingboth a cellulose portion and a nucleic acid portion, for exampleDNA-cellulose. In other embodiments, the composite polymer can include apolyacrylamide linked to a polynucleotide, and/or to a polypeptide.Inclusion of such composite polymers in the reaction mixture can beuseful in further retarding the movement of target polynucleotidesthrough the reaction mixture.

In some embodiments, the sieving agent can include polymers that havefirst been contacted or reacted with appropriate crosslinking agents.For example, the sieving agent can include acrylamide that has beenreacted with bis-acrylamide and/or Bis(acryloyl)cystamine.

In some embodiments, the reaction mixture includes at least onediffusion-reducing agent. In some embodiments, a diffusion-reducingagent comprises any compound that reduces migration of polynucleotidesfrom a region of higher concentration to one having a lowerconcentration. In some embodiments, a diffusion reducing agent comprisesany compound that reduces migration of any component of a nucleic acidamplification reaction irrespective of size. In some embodiments,components of a nucleic acid amplification reaction includebeads/primers, polynucleotides, recombinase, polymerase, nucleotides,ATP and/or co-factors.

It should be noted that the concepts of a sieving agent and adiffusion-reducing agent are not necessarily mutually exclusive; asieving agent can frequently be effective in reducing diffusion oftarget compounds through a reaction mixture, whereas a diffusionreducing agent can frequently have a sieving effect on reactioncomponents. In some embodiments, the same compound or reaction mixtureadditive can act both as a sieving agent and/or a diffusion reducingagent. Any of the sieving agents disclosed herein can in someembodiments be capable of acting as a diffusion reducing agent and viceversa.

In some embodiments, the diffusion reducing agent and/or sieving agentincludes polyacrylamide, agar, agarose or a cellulose polymer such ashydroxyethylcellulose (HEC), methyl-cellulose (MC) orcarboxymethylcellulose (CMC).

As described herein, polyclonal amplification can result fromamplification of multiple different polynucleotide templates within thesame continuous reaction mixture, optionally onto the same support orsurface. Unless the amplification reactions of different templates aresegregated or compartmentalized, polynucleotides within the reactionmixture can migrate via diffusion and/or Brownian motion during theamplification reaction, thus increasing the incidence of polyclonalamplification. Without being bound to any particular theory, in oneexemplary embodiment, polyclonal amplification results from themigration of one type of polynucleotide or its amplification productsfrom its initial location (e.g., a support or surface) to anotherlocation (e.g., another support or surface) via the reaction mixture.The other location can also contain polynucleotides of a different type,and hence the amplification of non-identical polynucleotide templates anon-monoclonal population can arise in the other location.

In some embodiments, the disclosure provides methods for providingsubstantially monoclonal amplification of multiple polynucleotidetemplates in parallel in a single continuous liquid phase of a reactionmixture, without need for compartmentalization or immobilization ofmultiple reactions. A suitable technique to reduce the production ofpolyclonal populations in certain embodiments involves increasing theaverage distance between the individual polynucleotide templates to beamplified within the same reaction mixture. Another suitable techniqueto reduce the production of polyclonal populations involves increasingthe volume of the reaction mixture. It is understood that applyingsuitable techniques, such as those described herein, can result in bothincreasing the volume of the reaction mixture and increasing the averagedistance between individual polynucleotide templates to be amplified.

In some embodiments, the average distance between polynucleotidetemplates within the reaction mixture and/or the volume of the reactionmixture can be increased by the addition of additional components to thereaction mixture. In some embodiments, the additional components caninclude discrete physical components, such as particles, beads,supports, and the like. In some embodiments, the discrete physicalcomponents can be useful in maintaining the separation between twodifferent polynucleotide templates to be amplified. In some embodiments,the discrete physical components can be useful in increasing theeffective distance between two polynucleotide templates to be amplified.“Effective distance”, as used herein, refers to the average path lengthtaken by a first polynucleotide to travel from its current location tothe location of a second polynucleotide within the same reactionmixture.

In some embodiments, these physical components can be made of anysuitable material, such as glass, silica, organic polymers (e.g.,polyacrylamides, agaroses, celluloses, polyalkylenes, polyaryls, etc.),metals, or metal or semi-metal oxides (e.g. aluminum oxides, titaniumoxides, zirconium oxides, silicon oxides, etc.). In some embodiments,the component can be formed from more than one component, such ascombinations of polymers, co-polymers, glass-polymer combinations,glasses, silica, metals, metal oxides or semi-metal oxides. In someembodiments, the components can include a glass that is passivated bythe polymer. In some embodiments, the components can include a soda-limeglass that is passivated by polyacrylamide (e.g.,poly-N,N-dimethylacrylamide). In some embodiments, the discrete physicalcomponents can be porous, wherein the porosity acts to limit, reduce orprevent migration of polynucleotides through the component. In someembodiments, the discrete physical components include cross-linkedpolymers. In some embodiments, the cross-linked polymers confer aporosity to the component, wherein the porosity acts to limit, reduce orprevent migration of polynucleotides through the component. In someembodiments, the additional components include a cationic orpolycationic molecule, such as quaternary amines, polylysine, or thelike, that can bind to the polyanionic polynucleotides.

In some embodiments, the additional components in the reaction mixturecan bind to migrating or diffusible polynucleotides. In someembodiments, the additional components can bind non-specifically tomigrating or diffusible polynucleotides. In some embodiments, theadditional components can bind to one or more specific nucleotidesequences on at least a portion of the migrating or diffusiblepolynucleotides. In some embodiments, the sequence-specificity of thenucleotide sequence binding of the additional components is provided byone or more oligonucleotide primers attached to the additionalcomponents, where the oligonucleotide primers include sequences that arecomplementary to at least a portion of the specific sequence. In someembodiments, the oligonucleotide primers attached to or otherwiseassociated with the additional components are random or degenerateoligonucleotide primers, such that the random or degenerate primers areable to bind a plurality of complementary sequence portions on migratingor diffusible polynucleotides. In some embodiments, the oligonucleotideprimers attached to the additional components are incapable oftemplate-directed elongation, such as by modification of the 3′-hydroxylgroup on the 3′ terminal nucleotide of the oligonucleotides. In thismanner, the extension-incapable primer can bind to migrating ordiffusible polynucleotides, either in a sequence-specific orsequence-non-specific manner, but because they are not capable offurther template-directed elongation they will not contribute to furtheramplification that may lead to polyclonality. In some embodiments, the3′ terminal nucleotide is a dideoxy nucleotide. In some embodimentsthese elongation-incompetent primers are modified, such as by modifiedinternucleotide linkages (e.g., thiophosphate) or blocking groups, so asto be resistant to exonucleases or proof-reading, such as 3′-5′exonuclease activity.

In some embodiments, the average distance between polynucleotidetemplates within the reaction mixture and/or the volume of theamplification reaction mixture can be implemented by introducing into orincreasing the amount of encapsulated gases or liquids in to thereaction mixture that are mostly immiscible with the reaction mixture.For example, in some embodiments, these encapsulated gases can be in theform of foam or bubbles within the reaction mixture. The foam or bubblescan contain air or other suitable gases. In some embodiments, thesuitable gas is a substantially inert gas such as nitrogen or helium. Insome embodiments, the encapsulated liquids can include a liquid that issubstantially immiscible with the reaction mixture.

For example, in some embodiments, the locations of differentpolynucleotide templates to be monoclonally amplified can be located ondiscrete solid supports, such as by immobilization or attachment tobeads or particles. In such embodiments, each discrete support elementcontains a substantially monoclonal polynucleotide population (typicallybut not necessarily a single polynucleotide template), and thus barringany cross-contamination by different migrating polynucleotides,amplification of the polynucleotides also results in a substantiallymonoclonal population linked to a discrete support.

Without intending to be bound by any particular model or theory, byadding the additional components described above to the amplificationreaction mixture, the additional components serve to surround andseparate the discrete polynucleotide templates from each other, therebyincreasing the distance between the individual templates, and thusincreasing the distance that migrating polynucleotides must traverse toencounter another template location, which may result in polyclonality.In some embodiments, the presence of the additional components may alsophysically impede or block the migration of polynucleotides within thereaction mixture, as they are typically impermeable to migratingpolynucleotides. In some embodiments, the additional components areessentially impermeable to the migrating polynucleotide as thecomponents are of a different phase of matter (e.g. solid or gas) thanthe reaction mixture (e.g. liquid).

In some embodiments, the addition of additional components to thereaction mixture can serve to reduce or eliminate polynucleotidemigration and polyclonality in small volumes of reaction mixtures. Inparticular, reducing the volume of the amplification reaction mixturemay be desirable in certain embodiments as it can improve efficiency ofthe reaction, or reduce the amount of needed reagents, or reduce thesize of the reaction vessel, or other advantages or any combinationthereof. However, reducing the volume may also decrease the distancebetween different polynucleotide templates in the reaction mixture,thereby potentially increasing the problem of polynucleotide migrationand hence polyclonality. Thus, in some embodiments, by also introducingthe additional components as described above, the migration ofpolynucleotides that may cause polyclonality can be reduced, mitigatedor prevented despite the small reaction volume as described herein.

In some embodiments, the addition of additional components describedabove to the reaction mixture, which contains the differentpolynucleotide templates to be amplified, serves to increase the totalvolume of the reaction volume. In such embodiments, and withoutintending to be bound by any model or theory, increasing the volume mayincrease the mean distance between any two or more differentpolynucleotide templates in the reaction mixture. In this manner, byincreasing the mean distance, the likelihood of migratingpolynucleotides traversing the distance between different polynucleotidetemplates, and thus reduce, mitigate or prevent polyclonality fromamplification of different polynucleotides at a given amplificationlocation.

In some embodiments, undesired polyclonality at a given polynucleotideamplification location can be reduced or substantially eliminated byseparating, segregating or isolating (either partially or completely)discrete amplification products from the bulk of the other amplificationproducts or other polynucleotide templates, particularly from thosehaving different polynucleotides. In particular, in some embodiments thediscrete amplification products to be separated have larger amounts ofpolynucleotides, such as would result from the monoclonal amplificationof polynucleotide templates at that location. Thus, separation,segregation or isolation can be effected based on the increased amountor density of nucleic acids at that location. In some embodiments, theseparation, segregation or isolation can be effected by subjecting thereaction mixture to an electric field, which would confer anelectromotive force on the polyanionic nucleic acids in a manner similarto electrophoresis. Thus, amplification locations having a higheranionic charge amount or density, such as those having increased amountsof amplified polynucleotides, would be separated by an electromotiveforce proportional to the amount of polynucleotide. Moreover, as theamount of amplified polynucleotide increases at a given location, theforce imposed by the electric field will increase, thereby furtherseparating the amplified polynucleotides. In some embodiments, differentpolynucleotide templates are each separately associated with discretesolid supports, such as beads or particles, which are contained in thereaction mixture. In these embodiments, polynucleotide amplificationproducts at discrete locations are capable of mobility within thereaction mixture when impelled by a suitable force, such as by theelectric field. In some embodiments, the electric field can be appliedto the reaction mixture continuously or transiently. In someembodiments, the electric field can be applied with a constant magnitudeor a varying magnitude. In some embodiments, the electric field can beapplied with a constant direction or a varying direction. In someembodiments, both the magnitude and the direction can be varied, eitheralternatively or concurrently, or a combination of both.

In some embodiments, the sieving agent and/or the diffusion reducingagent is included in the reaction mixture at concentrations of at least1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 74%, 90%, or 95% w/v(weight of agent per unit volume of reaction mixture).

In some embodiments, the reaction mixture includes at least one crowdingagent. For example, a crowding agent can increase the concentration ofone or more components in a nucleic acid amplification reaction bygenerating a crowded reaction environment. In some embodiments, thereaction mixture includes both a sieving agent and a crowding agent.

In some embodiments, the nucleic acid amplification includes agentscomprising aggregated or polymerized monomers, particularly monomersthat are suitable for gel electrophoretic separation of biomolecules,including separation of nucleic acids, peptides or saccharides.

In some embodiments, the agent can be used to prepare a gel or matrixunder conditions that produce gels or matrices having pores small enoughto retard movement of a given polynucleotide through the gel.

In some embodiments, the agent includes at least one polymer derivedfrom algae, plants, bacteria, fish or animals, or derivatives of thesepolymers. Optionally, the polymer includes linear, branched or highlybranched forms. Optionally, the polymer includes a glucose-basedpolymer, including cellulose, dextran, or starch. Optionally, thepolymer includes a galactose-based polymer, including hemicelluloses.Optionally, the polymer includes Ficoll. Optionally, the polymerincludes gellan gum. Optionally, the polymer includes agar, agarose oralginic acid. Optionally, the polymer includes copolymers or blends,including Phytagel™ or Agargel™. Optionally, the pore size in the gelcan be modulated by changing the pH, temperature, or concentration ofthe agent.

In some embodiments, the agent includes polymerized monomers of at leastone type of acrylamide or derivatives thereof, that are suitable for gelelectrophoretic separation of biomolecules, including nucleic acids,peptides or saccharides. Optionally, the agent includes at least onetype of cross-linking agent, including N,N′-methylene-bis-acrylamide(bis-acrylamide) or derivatives thereof. Optionally, the polymerizationreaction can be initiated by addition of a free radical source,including persulfate ions (e.g., from TEMED, tetramethylethylenediamine)or riboflavin. Optionally, the pore size in the gel can be modulated bychanges in pH, temperature, or concentration of acrylamide (orderivative) or cross-linking agent, or by addition of a compound thatinhibits the polymerization reaction, including Tris, borate, acetate,glycine, SDS or urea.

In some embodiments, the agent includes at least one oligomer or polymerof ethylene oxide suitable to prepare a hydrogel, including polyethyleneoxide (PEO), polyoxyethylene (POE), or polyethylene glycol (PEG).Optionally, the polymers of ethylene oxide can have branched, star orcomb structures. Optionally, the polymers of ethylene oxide can have anaverage molecular weight of about 200 to about 8000. Optionally,polymerization can be initiated by addition of a monofunctional methylether PEG or methoxypoly(ethylene glycol) (e.g., mPEG).

In some embodiments, the nucleic acid amplification includes agentscomprising polymerized monomers that are suitable for capillary gelelectrophoresis. In some embodiments, the monomers comprise acrylamideor derivatives thereof, including a linear dimethylacrylamide, orpoly(N,N-dimethylacrylamide)(e.g., pDMA). In some embodiments, the agentincludes at least one nucleic acid denaturant, such as urea or2-pyrrolidinone. Optionally, the agent includes about 0.1% to about 6%acrylamide, dimethylacrylamide, or poly(N,N-dimethylacrylamide).Optionally, the agent comprises about 0.1 M to about 9 M urea.Optionally, the agent includes about 0.1% to about 8% 2-pyrrolidinone.Optionally, the agent includes a POP polymer including POP-4, POP-5,POP-6, or POP-7 (Life Technologies, Carlsbad, Calif., USA)

In some embodiments, the nucleic acid amplification includes agents thatinclude a hydrogel. In some embodiments, the hydrogel includes apolymerizable monomer, including a hydrophilic monomer or a vinyl-basedmonomer. In some embodiments, the hydrogel includes monomers ofacrylamide or acrylamide derivatized to include hydroxyl groups, aminogroups, carboxyl groups, halogen groups, or any combination thereof.Optionally, the hydrogel includes monomers of aminoalkyl acrylamide oracrylopiperazine, or a combination thereof. Optionally, the hydrogelincludes monomers of hydroxyalkyl acrylamide, such as hydroxyethylacrylamide. Optionally, the hydroxyalkyl acrylamide can includeN-tris(hydroxymethyl)methyl)acrylamide, N-(hydroxymethyl)acrylamide, ora combination thereof. Optionally, the hydrogel includes a comonomer,including a halogen modified acrylate or acrylamide, such as aN-(5-bromoacetamidylpentyl)acrylamide (BRAPA). Optionally, the comonomerincludes an oligonucleotide modified acrylate or acrylamide monomer.Optionally, the hydrogel includes a mixture of monomers, includinghydroxyalky acrylamide and amine functionalize acrylamide, or a mixtureof acrylamide and amine functionalized acrylamide.

In some embodiments, the nucleic acid amplification includes an agentthat can form a gel having a network of polymers joined by chemicalbonds or physical associations to form a reversible or irreversible gel.In some embodiments, an irreversible gel includes irreversible chemicalbonds. In some embodiments, a reversible gel includes reversiblechemical bonds or reversible physical associations. In some embodiments,the polymers in the network can be joined by physical associations thatinclude hydrophobic interactions, hydrophilic interactions, hydrogenbonds, metal coordination or electrostatic interactions. In someembodiments, the chemical bonds or physical associations between thepolymers in the network can be reversed to convert the gel into aliquid-like state by introducing a stimulus, including pH, temperature,pressure, ultrasound, magnetic field, metal chelation, mechanical force,solvent composition, ionic strength, reducing agent, photoelectricstimulation or electromagnetic radiation. Optionally, a reversible gelincludes at least one naturally-occurring or synthetic polymer includinggellan gum, aginate compound (e.g., agarose), cellulose or cellulosederivatives, dextran, glycomannan, polyethylene glycol, nonioinictriblock copolymers (e.g., Poloxamers' or Pluronics™), acrylamide,poly-N-isopropylacrylamide (e.g., poly-NIPA),poly-N,N-dimethylacrylamide (e.g., DMA/DEA), N-alkylacrylamides,N-isopropylacrylamide, dimethylaminoethyl methyacrylate (e.g., DMAEMA),2-hydroxyethyl methacrylate (e.g., HEMA), poly(N-isopropylacrylamide)(e.g., PNIPAm). Optionally, the reversible gel includes acrylamide, oran acrylamide derivative, with a bis-acrylamide cross-linker replacedwith a nucleic acid cross-linker. Optionally, the nucleic acidcross-linker includes DNA or DNA-based nanodevices, with or withoutacrydite modification. Optionally, the reversible gel includespolyacrylamide with thiol-bridge cross-linkages (e.g., di-thiolacrylamide). Optionally, the thiol cross linked acrylamide gel can beconverted to a soft-gel or liquid-like state by adding a reducing agentincluding DTT (dithiothreitol) or TCEP (Tris(2-carboxyethyl)phosphinehydrochloride).

In some embodiments, the nucleic acid amplification includes agentscomprising a polymer functionalized with at least one type of aromaticmoiety. In some embodiments, the polymer includes acrylamide or aderivative thereof. In some embodiments, the aromatic moiety can bindnon-specifically to nucleic acids, including for example bind the basesin single-stranded or double-stranded nucleic acid molecules. In someembodiments, a polymer matrix (e.g., acrylamide) with aromatic moietiesforms a “sticky” web that binds nucleic acids and slows migration of thenucleic acids through the reaction mixture. Optionally, the aromaticmoiety includes benzene, or derivatives including toluene, phenol,aniline, acetophenone, benzaldehyde, benzoic acid, benzonitrile,ortho-xylene, styrene, or an aromatic amino acid (e.g., histidine,phenylalanine, or tryptophan).

In some embodiments, the nucleic acid amplification includes one or moreagents suitable for preparing an electrophoretic hydrogel. In someembodiments, the agent includes at least one polymer and one or morecapture moieties that bind at least a portion of a nucleic acid moleculein the amplification reaction, including single-stranded ordouble-stranded nucleic acid templates or primers. In some embodiments,the capture moiety can bind the nucleic acid and retard the movement ofthe nucleic acid in the amplification reaction. In some embodiments, thecapture moiety can be bound or associated with the polymer, or thecapture moiety is not associated with the polymer. In some embodiments,the capture moiety can be placed in a fixed position within theamplification reaction, or can be located throughout the amplificationreaction. In some embodiments, the capture moiety can be placed on asupport, including a bead or reaction chamber. In some embodiments, thecapture moiety can bind specifically or non-specifically to the nucleicacids. In some embodiments, the capture moiety can bind the nucleicacids by covalent interaction, or by non-covalent interaction includingionic bonds, hydrophobic interactions, hydrogen bonds, van der Waalsforces or dipole-dipole interactions. Optionally, the polymer includes apore-size gradient or lacks a gradient. Optionally, the polymer includesagarose, or polyacrylamide or derivatives thereof including acrylatepolymers, alkylacrylate polymers, or alkyl alkylacrylate polymers.Optionally, the polymer includes at least one type of polymerizedcomonomer includingN-(3-[(4-benzoylphenyl)formamido]propyl)methacrylamide (e.g., BPMA orBPMAC). Optionally, the capture moieties comprise any compound thatbinds nucleic acids, including hybridizable nucleic acids or one memberof a binding partner where the other member is joined to the nuclei acidin the amplification reaction. Optionally, the capture moiety binds thenucleic acids in the amplification reaction upon applying at least onestimulus. Optionally, the stimulus includes pH, temperature, pressure,ultrasound, magnetic field, metal chelation, mechanical force, solventcomposition, ionic strength, photoelectric stimulation or radiation.Optionally, the nucleic acid amplification reactions can be conductedwith polymers and capture moieties that are suitable for preparingelectrophoretic hydrogels as described by Amy Herr (see published U.S.patent application Nos. 2011/0177618, 2012/0329040, 2012/0142904,2012/0135541, and 2013/0078663).

In some embodiments, the disclosure relates generally to methods, aswell as related compositions, systems, kits and apparatuses, for liquidhandling to perform nucleic acid amplification methods.

In various applications relating to liquid handling, for example in thefield of microfluidics, electrowetting has been used to manipulateliquid behavior. Electrowetting involves the use of an electric field toalter the wetting behavior of liquid relative to a surface so as tocontrol the movement of the liquid. In other words, through theapplication of an electric potential, a liquid-solid interface can bealtered by controlling the wettability of the surface (e.g., effectivelyconverting the surface in contact with the liquid from hydrophobic tohydrophilic or vice versa) to thereby control movement of a liquid onthat surface.

In some embodiments, electrowetting can be used to precisely divideand/or position liquid, without the need to utilize pumps, valves,channels, and/or other similar fluid handling mechanisms. As an example,electrowetting may include sandwiching the liquid between two supports(e.g., plates) and in contact with an insulated electrode. By applyingan electric field in a non-uniform manner so as to create a surfaceenergy gradient, a large number of small volumes of liquid (e.g.,droplets, beads, cells, or other small volumes) can be independentlymanipulated under direct electrical control. In some embodiments, thesupport includes electrodes arranged in any pattern, including one ormore paths or arrays. In some embodiments, the electrodes may or may notbe in direct contact with the fluid. In some embodiments, the electrodesmay be configured such that the support has a hydrophilic side and ahydrophobic side. In some embodiments, the droplets subjected to thevoltage will move towards the hydrophilic side. In some embodiments, thepattern of electrodes may be a high density pattern. See, for example,“Electrowetting-Based On-Chip Sample Processing for IntegratedMicrofluidics,” Electron Devices Meeting, 2003. IEDM '03 TechnicalDigest. IEEE International, pages 32.5.1-32.5.4; R. B. Fair, V.Srinivasan, H. Ren, P. Paik, V. K. Pamula, M. G. Pollack.

In some embodiments, electrowetting may include using an electricvoltage to alter the shape of a liquid droplet. In some embodiments,electrowetting may involve a sessile droplet positioned on adielectric-coated electrode. In some embodiments, when current isapplied, the drop flattens and flows out to the sides, thereby wettingadditional surface. When current is removed, the drop returns to itsoriginal shape and retracts from the areas covered upon currentapplication. See, for example, “New Methods to Transport Fluids inMicro-Sized Devices,” Lincoln Laboratory Journal, Volume 17, Number 2,(2008); S. Berry and J. Kedzierski.

In some embodiments, the use of electrowetting in the field of liquidhandling for biological reactions and/or analyses may provide relativelyaccurate and fast manipulation of a large number of small volumes ofliquid. For example, liquid containing reagents for performing abiological reaction (e.g., assays, testing, and other relatedprocedures) can be dispensed into numerous small reservoirs, such aswells in titer plates, for example, with a compact device (e.g., loader)that provides both liquid handling (e.g., positioning) and dispensing.Such a device may replace a liquid handling robot or be incorporatedwithin a biological analysis workstation. In some embodiments, loadingconfiguration of the dispensed liquid may be programmed by computer,e.g. a 96-, 384-, 768-, 1536-, 3072-, 6144-, 12,288-, or 24,576-wellformat. In some embodiments, the amount of liquid dispensed may includedroplets, cells, beads, or other amounts, in an exact number (e.g., theamount of liquid dispensed may be controlled). Moreover, the preciselocations to which the liquid is dispensed may be controlled.

In some embodiments, liquid handling can be performed using adiscrete-flow device that can form and move individual liquid droplets.In some embodiments, the discrete flow device comprises a dropletmicroactuator. In some embodiments, the droplet microactuator comprisesone or more substrates configured to form a surface. In someembodiments, the surface includes one or more gaps or channels. In someembodiments, the droplet microactuator moves a droplet from one positionto a different position on the substrate, or within the gap or channel.

In some embodiments, the substrates can be associated with electrodesthat are arranged in any pattern, including at least one path or array,to provide an electric field for forming or moving the droplet. In someembodiments, the droplet microactuator comprises an electrowettingdevice (Pollack 2000 Applied Physics Letters 77:1725-1727). In someembodiments, the electrodes can be disposed on the substrate, or in thegap or along the channel. In some embodiments, the droplet comprises anaqueous liquid. In some embodiments, the substrates can be coated with,or the gap or channel between the substrates can be filled with, aliquid that is immiscible with the liquid that forms the droplets. Insome embodiments, the immiscible liquid comprises an oil, including asilicone oil, fluorosilicone oil, or hexadecane oil. In someembodiments, the immiscible liquid comprises at least one surfactant. Insome embodiments, the aqueous droplet can be in contact with, surroundedby, or floating in, the immiscible liquid. In some embodiments, theaqueous droplet and an immiscible liquid can be sandwiched between twosubstrates that are separated to form a gap. In some embodiments, theaqueous droplet can form and move within the gap. In some embodiments,the gap can be elongated to form a channel or path. In some embodiments,the electric field can be applied to one area of the substrate whichlowers the interfacial tension in that area, causing a nearby droplet tomove towards the location of the activated electrode, therebytransporting the droplet from one location to another location on thesubstrate (U.S. published application Nos. 2013/0203606, 2013/0225452and 2013/0225450, and U.S. Pat. No. 7,851,184).

In some embodiments, the droplet microactuator can perform at least onedroplet operation including: loading a droplet into the dropletmicroactuator; dispensing at least one droplet from a source or a sourcedroplet; transporting a droplet from one location to another in anydirection in the droplet microactuator; merging two or more dropletsinto a single droplet; mixing a droplet; splitting a droplet into two ormore droplets; diluting a droplet; agitating a droplet; deforming adroplet; retaining a droplet in a position; incubating a droplet;heating a droplet; cooling a droplet; vaporizing a droplet; disposing adroplet; transporting a droplet out of a droplet actuator; or anycombination of these droplet operations.

In some embodiments, the droplets can include minute volumes of liquid,including about 1×10⁻¹² liters to about 1×10⁻³ liters, or about 10⁻⁹liters to about 10⁻⁶ liters.

In some embodiments, the droplets can have any shape includingspherical, truncated sphere, disc, slug, ellipsoid, ovoid, cylindrical,or any combination of these shapes.

In some embodiments, at least one liquid droplet (e.g., aqueous droplet)containing one or more nucleic acid amplification reagents can be movedfrom one position to a different position on a substrate. In someembodiments, movement of the droplets on the substrate can be used toconduct a nucleic acid amplification reaction. In some embodiments, thesubstrate comprises a droplet microactuator. In some embodiments, thedroplet microactuator forms one or more liquid droplets containing atleast one nucleic acid amplification reagent, and moves the droplet fromone position to a different position on the droplet microactuator (e.g.,performs at least one droplet operation).

Optionally, individual droplets can contain the same or differentnucleic acid amplification reagents.

Optionally, individual droplets can contain one or more polynucleotidetemplates.

Optionally, different droplets can contain polynucleotide templateshaving the same or different sequences.

Optionally, one or more nucleic acid amplification reagents can beattached to the substrate, or attached to a gap or channel in thesubstrate, in any shape or pattern, including a region, spot, path orarray.

Optionally, one or more nucleic acids, including a polynucleotidetemplate or capture oligonucleotide, can be attached to the substrate.

Optionally, individual regions, spots, or paths or different positionsin the arrays, can be attached to at least one nucleic acid or can beattached to different nucleic acids having the same sequence or havingtwo or more different sequences.

Optionally, an affinity moiety (e.g., avidin or avidin-like compound)can be attached to the substrate.

Optionally, the substrate includes a surface that is configured with aheating and/or cooling source for modulating the temperature of adroplet.

Optionally, the substrate includes a surface that is configured with amagnetic field source for applying a magnetic force to the contents of adroplet.

In some embodiments, reagents for conducting a nucleic acidamplification reaction include any one or more of the following: atleast one support (e.g., a bead or particle), one or more polynucleotidetemplates, recombinase, polymerase, oligonucleotide primers (e.g.,first, second, third, or additional oligonucleotide primers),nucleotides, at least one divalent cations (e.g., magnesium and/ormanganese), ATP, co-factors, sieving agents, diffusion reducing agentsand accessory proteins (e.g., helicase, single-stranded binding proteinsand recombinase loading factor).

Optionally, the droplet microactuator can form at least one droplet thatcontains all of the reagents necessary for conducting a nucleic acidamplification reaction. Optionally, the at least one droplet comprisesat least one support (e.g., a bead or particle), one or morepolynucleotide templates, recombinase, polymerase, oligonucleotideprimers (e.g., first, second, third, or additional oligonucleotideprimers), nucleotides, at least one divalent cations (e.g., magnesiumand/or manganese), ATP, co-factors, and accessory proteins (e.g.,helicase, single-stranded binding proteins and recombinase loadingfactor). Optionally, the droplet microactuator can transport the atleast one droplet to a location in the droplet microactuator having aheating or cooling source for incubating the droplet at a temperaturethat is suitable for conducting the nucleic acid amplification reaction.

In some embodiments, the disclosure relates generally to methods,systems, kits, apparatuses and compositions for forming a plurality ofdroplets, wherein at least two droplets of the plurality eachindependently contains a single polynucleotide template. Optionally, theat least two droplets include one or more reagents useful for nucleicacid amplification. Alternatively, the at least two droplets can beindividually fused, coalesced or merged with at least one other dropletincluding one or more reagents useful for nucleic acid amplification.The one or more reagents optionally include any one or more of thefollowing: polymerase, nucleotides, recombinase, accessory factors,primers, buffer, salts, and the like. In some embodiments, once two ormore droplets are formed that include individual nucleic acid templatesand reagents for nucleic acid amplification, the droplets can besubjected to amplification conditions. The amplification conditionsoptionally include any of the amplification conditions disclosed herein.Optionally, the individually distributed templates within the dropletsare clonally amplified within their respective droplets, resulting inthe formation of at least two droplets each including a substantiallymonoclonal nucleic acid population.

Optionally, the droplet microactuator can split a droplet into multipledroplets. Optionally, the droplet microactuator can form at least onedroplet that contains all or a subset of the reagents for conductingnucleic acid amplification reactions, and split the droplet into two ormore droplets. Optionally, individual droplets from the multipledroplets contain the same reagents or different reagents. Optionally,individual droplets from the multiple droplets contain the same ordifferent polynucleotide templates. Optionally, at least one of theindividual droplets contains all reagents for conducting a nucleic acidamplification reaction, including at least one support (e.g., a bead orparticle), one or more polynucleotide templates, recombinase,polymerase, oligonucleotide primers (e.g., first, second, third, oradditional oligonucleotide primers), nucleotides, at least one divalentcations (e.g., magnesium and/or manganese), ATP, co-factors andaccessory proteins (e.g., helicase, single-stranded binding proteins andrecombinase loading factor). Optionally, the droplet microactuator cantransport at least one of the multiple droplets to a location in thedroplet microactuator having a heating or cooling source for incubatingthe droplets at a temperature that is suitable for conducting thenucleic acid amplification reaction. Optionally, individual dropletscontaining one or a plurality of polynucleotide templates having thesame sequence can produce a monoclonal population of the amplificationproducts attached to one or more supports (e.g., beads or particles).Optionally, individual droplets containing multiple polynucleotidetemplates having different sequences, can produce a polyclonalpopulation of the amplification products attached to supports (e.g.,beads or particles).

Optionally, the droplet microactuator can be used to amplify nucleicacids and attach the amplification products to the substrate.Optionally, the droplet microactuator comprises a substrate attached toat least one oligonucleotide primer (e.g., a first primer). The positionon the substrate that is attached to the first primer can optionallyinclude a heating or cooling source. Optionally, the dropletmicroactuator can form at least one droplet containing one or morepolynucleotide templates, recombinase, polymerase, second and thirdoligonucleotide primers (and optionally other primers), nucleotides, atleast one divalent cations (e.g., magnesium and/or manganese). ATP,co-factors, and accessory proteins (e.g., helicase, single-strandedbinding proteins and recombinase loading factor). Optionally, the firstprimer can hybridize to at least a portion of the polynucleotidetemplate. Optionally, the droplet microactuator can move the at leastone droplet from a first position on the substrate to the position onthe substrate that is attached with the first primers, to permithybridization between the immobilized first primers and thepolynucleotide templates, and form a droplet containing all of thereagents necessary for conducting the nucleic acid amplificationreaction. Optionally, the heating or cooling source can be activated toproduce a temperature suitable for performing the nucleic acidamplification reaction.

Optionally, the nucleic acid amplification reagents can be split intotwo or more separate droplets so that the amplification reaction doesnot occur until the droplets are merged. Optionally, the dropletmicroactuator can form a first droplet containing at least one divalentcation (e.g., magnesium and/or manganese), and a second dropletcontaining the remaining reagents for conducting a nucleic acidamplification reaction. Optionally, the second droplet comprises atleast one support (e.g., a bead or particle), one or morepolynucleotides, recombinase, polymerase, oligonucleotide primers (e.g.,first, second, third, or additional oligonucleotide primers),nucleotides, ATP, co-factors and accessory proteins (e.g., helicase,single-stranded binding proteins and recombinase loading factor).Optionally, the droplet microactuator can merge the first and the seconddroplets to form a single droplet containing all of the reagents forconcluding a nucleic acid amplification reaction. Optionally, thedroplet microactuator can merge the two droplets by transporting thefirst droplet to the location of the second droplet, or vice versa.Optionally, the droplet microactuator can transport, the merged dropletto a location in the droplet microactuator having a heating or coolingsource for incubating the merged droplets at a temperature that issuitable for conducting the nucleic acid amplification reaction.

Optionally, a droplet microactuator can be used to enrich nucleic addamplification products in a droplet. Optionally, a droplet microactuatorcan be used to perform a nucleic acid amplification reaction in thepresence of one member of a binding partner (e.g., biotin). Optionally,the droplet microactuator can form at least one droplet comprising atleast one support (e.g., a bead or particle), one or more polynucleotidetemplates, recombinase, polymerase, oligonucleotide primers (e.g.,first, second, third, or additional oligonucleotide primers),nucleotides, at least one divalent cation (e.g., magnesium and/ormanganese), ATP, co-factors, accessory proteins (e.g., helicase,single-stranded binding proteins and recombinase loading factor) andbiotin. Optionally, the biotin can be attached to at least oneoligonucleotide primer. Optionally, a droplet microactuator can incubatethe at least one droplet under conditions that are suitable forgenerating nucleic acid amplification products that include the onemember of a binding partner (e.g., biotin). Optionally, the other memberof the binding partner avidin or avidin-like compound) can be attachedto a location on the droplet microactuator. Optionally, the dropletmicroactuator can transport the at least one droplet to the locationhaving the other member of the binding partner, to permit bindingbetween the two members of the binding partners. Optionally, the dropletmicroactuator can remove some of the liquid from the droplet therebyremoving reagents that are not bound to the binding partners.

Optionally, a droplet microactuator can be used to enrich magneticsupports. Optionally, a droplet microactuator can include a surface thatis configured to apply a magnetic field. Optionally, the dropletmicroactuator can form at least one droplet comprising at least onemagnetic responsive support (e.g., beads or particles), one or morepolynucleotide templates, recombinase, polymerase, oligonucleotideprimers (e.g., first, second, third, or additional oligonucleotideprimers), nucleotides, at least one divalent cation (e.g., magnesiumand/or manganese), ATP, co-factors and accessory proteins (e.g.,helicase, single-stranded binding proteins and recombinase loadingfactor). Optionally, the droplet microactuator can transport the atleast one droplet to a first location in the droplet microactuatorhaving a heating or cooling source for incubating the droplet at atemperature that is suitable for generating nucleic acid amplificationproducts attached to the magnetic responsive supports. Optionally, thedroplet microactuator can transport at least one droplet to a secondlocation having the magnetic field to attract the magnetic-responsivesupports. Optionally, the droplet microactuator can remove some of theliquid from the droplet thereby removing reagents that are not attractedto the magnetic field or that are not attached to themagnetic-responsive support.

One skilled in the art will recognize that the droplet microactuator canform droplets having any combination or subcombination of nucleic acidamplification reaction reagents, and that the droplet microactuator canperform any combination or subcombination of droplet operations toconduct the nucleic acid amplification reaction.

In some embodiments, methods for nucleic acid amplification comprise oneor more surfaces. In some embodiments, a surface can be attached with aplurality of first primers, the first primers of the plurality sharing acommon first primer sequence.

In some embodiments, a surface can be an outer or top-most layer orboundary of an object. In some embodiments, a surface can be interior tothe boundary of an object.

In some embodiments, the reaction mixture includes multiple differentsurfaces, for example, the reaction mixture can include a plurality ofbeads (such as particles, nanoparticles, microparticles, and the like)and at least two different polynucleotide templates can be clonallyamplified onto different surfaces, thereby forming at least twodifferent surfaces, each of which is attached to an amplicon. In someembodiments, the reaction mixture includes a signal surface (forexample, the surface of a slide or array of reaction chambers) and atleast two different polynucleotide templates are amplified onto twodifferent regions or locations on the surface, thereby forming a singlesurface attached to two or more amplicons.

In some embodiments, a surface can be porous, semi-porous or non-porous.In some embodiments, a surface can be a planar surface, as well asconcave, convex, or any combination thereof. In some embodiments, asurface can be a bead, particle, microparticle, sphere, filter,flowcell, well, groove, channel reservoir, gel or inner wall of acapillary. In some embodiments, a surface includes the inner walls of acapillary, a channel, a well, groove, channel, reservoir. In someembodiments, a surface can include texture (e.g., etched, cavitated,pores, three-dimensional scaffolds or bumps).

In some embodiments, particles can have a shape that is spherical,hemispherical, cylindrical, barrel-shaped, toroidal, rod-like,disc-like, conical, triangular, cubical, polygonal, tubular, wire-likeor irregular.

In some embodiments, a surface can be made from any material, includingglass, borosilicate glass, silica, quartz, fused quartz, mica,polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate(PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS),silicon, germanium, graphite, ceramics, silicon, semiconductor, highrefractive index dielectrics, crystals, gels, polymers, or films (e.g.,films of gold, silver, aluminum, or diamond).

In some embodiments, a surface can be magnetic or paramagnetic bead(e.g., magnetic or paramagnetic nanoparticles or microparticles). Insome embodiments, paramagnetic microparticles can be paramagnetic beadsattached with streptavidin (e.g., Dynabeads™ M-270 from Invitrogen,Carlsbad, Calif.). Particles can have an iron core, or comprise ahydrogel or agarose (e.g., Sepharose™).

In some embodiments, the surface can be attached with a plurality of afirst primer. A surface can be coated with an acrylamide, carboxylic oramine compound for attaching a nucleic acid (e.g., a first primer). Insome embodiments, an amino-modified nucleic acid (e.g., primer) can beattached to a surface that is coated with a carboxylic acid. In someembodiments, an amino-modified nucleic acid can be reacted with EDC (orEDAC) for attachment to a carboxylic acid coated surface (with orwithout NHS). A first primer can be immobilized to an acrylamidecompound coating on a surface. Particles can be coated with anavidin-like compound (e.g., streptavidin) for binding biotinylatednucleic acids.

In some embodiments, the surface comprises the surface of a bead. Insome embodiments, a bead comprises a polymer material. For example, abead comprises a gel, hydrogel or acrylamide polymers. A bead can beporous. Particles can have cavitation or pores, or can includethree-dimensional scaffolds. In some embodiments, particles can be IonSphere™ particles.

In some embodiments, the disclosed methods (as well as relatedcompositions, systems and kits) include immobilizing one or more nucleicacid templates onto one or more supports. Nucleic acids may beimmobilized on the solid support by any method including but not limitedto physical adsorption, by ionic or covalent bond formation, orcombinations thereof. A solid support may include a polymeric, a glass,or a metallic material. Examples of solid supports include a membrane, aplanar surface, a microtiter plate, a bead, a filter, a test strip, aslide, a cover slip, and a test tube. means any solid phase materialupon which a oligomer is synthesized, attached, ligated or otherwiseimmobilized. A support can optionally comprise a “resin”, “phase”,“surface” and “support”. A support may be composed of organic polymerssuch as polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A support may also be inorganic, such as glass, silica,controlled-pore-glass (CPG), or reverse-phase silica. The configurationof a support may be in the form of beads, spheres, particles, granules,a gel, or a surface. Surfaces may be planar, substantially planar, ornon-planar. Supports may be porous or non-porous, and may have swellingor non-swelling characteristics. A support can be shaped to comprise oneor more wells, depressions or other containers, vessels, features orlocations. A plurality of supports may be configured in an array atvarious locations. A support is optionally addressable (e.g., forrobotic delivery of reagents), or by detection means including scanningby laser illumination and confocal or deflective light gathering. Anamplification support (e.g., a bead) can be placed within or on anothersupport (e.g., within a well of a second support).

In an embodiment the solid support is a “microparticle,” “bead”“microbead”, etc., (optionally but not necessarily spherical in shape)having a smallest cross-sectional length (e.g., diameter) of 50 micronsor less, preferably 10 microns or less, 3 microns or less, approximately1 micron or less, approximately 0.5 microns or less, e.g., approximately0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,about 1-10 nanometer, about 10-100 nanometers, or about 100-500nanometers). Microparticles (e.g., Dynabeads from Dynal, Oslo, Norway)may be made of a variety of inorganic or organic materials including,but not limited to, glass (e.g., controlled pore glass), silica,zirconia, cross-linked polystyrene, polyacrylate, polymehtymethacrylate,titanium dioxide, latex, polystyrene, etc. Magnetization can facilitatecollection and concentration of the microparticle-attached reagents(e.g., polynucleotides or ligases) after amplification, and can alsofacilitate additional steps (e.g., washes, reagent removal, etc.). Incertain embodiments of the invention a population of microparticleshaving different shapes sizes and/or colors can be used. Themicroparticles can optionally be encoded, e.g., with quantum dots suchthat each microparticle can be individually or uniquely identified.

In some embodiments, a bead surface can be functionalized for attachinga plurality of a first primer. In some embodiments, a bead can be anysize that can fit into a reaction chamber. For example, one bead can fitin a reaction chamber. In some embodiments more than one bead can fit ina reaction chamber. In some embodiments, the smallest cross-sectionallength of a bead (e.g., diameter) can be about 50 microns or less, orabout 10 microns or less, or about 3 microns or less, approximately 1micron or less, approximately 0.5 microns or less, e.g., approximately0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,about 1-10 nanometer, about 10-100 nanometers, or about 100-500nanometers).

In some embodiments, a bead can be attached with a plurality of one ormore different primer sequences. In some embodiments, a bead can beattached with a plurality of one primer sequence, or can be attached aplurality of two or more different primer sequences. In someembodiments, a bead can be attached with a plurality of at least 1,000primers, or about 1,000-10,000 primers, or about, 10,000-50,000 primers,or about 50,000-75,000 primers, or about 75,000-100,000 primers, ormore.

In some embodiments, the reaction mixture comprises a recombinase. Therecombinase can include any agent that is capable of inducing, orincreasing the frequency of occurrence, of a recombination event. Arecombination event includes any event whereby two differentpolynucleotides strands are recombined with each other. Recombinationcan include homologous recombination. The recombinase can be an enzyme,or a genetically engineered derivative thereof. The recombinaseoptionally can associate with (e.g., bind) a single-strandoligonucleotide (e.g., a first primer). In some embodiments, an enzymethat catalyzes homologous recombination can form a nucleoprotein complexby binding a single-stranded oligonucleotide. In some embodiments, ahomologous recombination enzyme, as part of a nucleoprotein complex, canbind a homologous portion of a double-stranded polynucleotide. In someembodiments, the homologous portion of the polynucleotide can hybridizeto at least a portion of the first primer. In some embodiment, thehomologous portion of the polynucleotide can be partially or completelycomplementary to at least a portion of the first primer.

In some embodiments, a homologous recombination enzyme can catalyzestrand invasion by forming a nucleoprotein complex and binding to ahomologous portion of a double-stranded polynucleotide to form arecombination intermediate having a triple-strand structure (D-loopformation) (U.S. Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos. 5,273,881and 5,670,316 both to Sena, and U.S. Pat. Nos. 7,270,981, 7,399,590,7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000, 8,062,850, and8,071,308). In some embodiments, a homologous recombination enzymecomprises wild-type, mutant, recombinant, fusion, or fragments thereof.In some embodiments, a homologous recombination enzyme comprises anenzyme from any organism, including myoviridae (e.g., uvsX frombacteriophage T4, RB69, and the like) Escherichia coli (e.g., recA), orhuman (e.g., RAD51).

In some embodiments, methods for nucleic acid amplification comprise oneor more accessory proteins. For example, an accessory protein canimprove the activity of a recombinase enzyme (U.S. Pat. No. 8,071,308granted to Piepenburg, et al.). In some embodiments, an accessoryprotein can bind single strands of nucleic acids, or can load arecombinase onto a nucleic acid. In some embodiments, an accessoryprotein comprises wild-type, mutant, recombinant, fusion, or fragmentsthereof. In some embodiments, accessory proteins can originate from anycombination of the same or different species as the recombinase enzymethat are used to conduct a nucleic acid amplification reaction.Accessory proteins can originate from any bacteriophage including amyoviral phage. Examples of a myoviral phage include T4, T2, T6, Rb69,Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophageP-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage25, Vibrio phage nt-1, phi-1, Rb16, Rb43, Phage 31, phage 44RR2.8t,Rb49, phage Rb3, and phage LZ2. Accessory proteins can originate fromany bacterial species, including Escherichia coli, Sulfolobus (e.g., S.solfataricus) or Methanococcus (e.g., M. jannaschii).

In some embodiments, methods for nucleic acid amplification can includesingle-stranded binding proteins. Single-stranded binding proteinsinclude myoviral gp32 (e.g., T4 or RB69), Sso SSB from Sulfolobussolfataricus, MjA SSB from Methanococcus jannaschii, or E. coli SSBprotein.

In some embodiments, methods for nucleic acid amplification can includeproteins that can improve recombinase loading onto a nucleic acid. Forexample, a recombinase loading protein comprises a UsvY protein (U.S.Pat. No. 8,071,308 granted to Piepenburg).

In some embodiments, methods for nucleic acid amplification can includeat least one proteins that bind nucleic acids, including proteins thatunwind duplex nucleic acids (e.g., helicase).

In some embodiments, methods for nucleic acid amplification can includeat least one co-factor for recombinase or polymerase activity. In someembodiments, a co-factor comprises one or more divalent cation. Examplesof divalent cations include magnesium, manganese and calcium.

In some embodiments, a nucleic acid amplification reaction can bepre-incubated under conditions that inhibit premature reactioninitiation. For example, one or more components of a nucleic acidamplification reaction can be withheld from a reaction vessel to preventpremature reaction initiation. To start the reaction, a divalent cationcan be added (e.g., magnesium or manganese). In another example, anucleic acid amplification reaction can be pre-incubated at atemperature that inhibits enzyme activity. The reaction can bepre-incubated at about 0-15° C., or about 15-25° C. to inhibit prematurereaction initiation. The reaction can then be incubated at a highertemperature to induce enzymatic activity.

In some embodiments, methods for nucleic acid amplification can includeat least one co-factor for recombinase assembly on nucleic acids or forhomologous nucleic acid pairing. In some embodiments, a co-factorcomprises any form of ATP including ATP and ATPγS.

In some embodiments, methods for nucleic acid amplification can includeat least one co-factor that regenerates ATP. For example, a co-factorcomprises an enzyme system that converts ADP to ATP. In someembodiments, a co-factor comprises phosphocreatine and creatine kinase.

In some embodiments, any of the nucleic acid amplification methodsdisclosed herein can be conducted, or can include steps that areconducted, under isothermal or substantially isothermal amplificationconditions. In some embodiments isothermal amplification conditionscomprise a nucleic acid amplification reaction subjected to atemperature variation which is constrained within a limited range duringat least some portion of the amplification (or the entire amplificationprocess), including for example a temperature variation is equal or lessthan about 20° C., or about 10° C., or about 5° C., or about 1-5° C., orabout 0.1-1° C., or less than about 0.1° C.

In some embodiments, an isothermal nucleic acid amplification reactioncan be conducted for about 2, 5, 10, 15, 20, 30, 40, 50, 60 or 120minutes.

In some embodiments, an isothermal nucleic acid amplification reactioncan be conducted at about 15-25° C., or about 25-35° C., or about 35-40°C., or about 40-45° C., or about 45-50° C., or about 50-55° C., or about55-60° C.

In some embodiments, methods for nucleic acid amplification comprise aplurality of different polynucleotides. In some embodiments, a pluralityof different polynucleotides comprises single-stranded ordouble-stranded polynucleotides, or a mixture of both. In someembodiments, a plurality of different polynucleotides comprisespolynucleotides having the same or different sequences. In someembodiments, a plurality of different polynucleotides comprisespolynucleotides having the same or different lengths. In someembodiments, a plurality of different polynucleotides comprises about2-10, or about 10-50, or about 50-100, or about 100-500, or about500-1,000, or about 1,000-5,000, or about 10³-10⁶, or about 10⁶-10¹⁰ ormore different polynucleotides. In some embodiments, a plurality ofdifferent polynucleotides comprises polymers of deoxyribonucleotides,ribonucleotides, and/or analogs thereof. In some embodiments, aplurality of different polynucleotides comprises naturally-occurring,synthetic, recombinant, cloned, amplified, unamplified or archived(e.g., preserved) forms. In some embodiments, a plurality of differentpolynucleotides comprises DNA, cDNA RNA or chimeric RNA/DNA, and nucleicacid analogs.

In some embodiments, a plurality of different polynucleotide templatescan comprise a double-stranded polynucleotide library construct havingone or both ends joined with a nucleic acid adaptor sequence. Forexample, a polynucleotide library construct can comprise a first andsecond end, where the first end is joined to a first nucleic acidadaptor. A polynucleotide library construct can also include a secondend joined to a second nucleic acid adaptor. The first and secondadaptors can have the same or different sequence. In some embodiments,at least a portion of the first or second nucleic acid adaptor (i.e., aspart of the polynucleotide library construct) can hybridize to the firstprimer. In some embodiments, a homologous recombination enzyme, as partof a nucleoprotein complex, can bind to a polynucleotide libraryconstruct having a first or second nucleic acid adaptor sequence.

In some embodiments, polynucleotide library constructs can be compatiblefor use in any type of sequencing platform including chemicaldegradation, chain-termination, sequence-by-synthesis, pyrophosphate,massively parallel, ion-sensitive, and single molecule platforms.

In some embodiments, methods for nucleic acid amplification includediluting the amount of polynucleotides that are reacted with beads(e.g., beads attached with a plurality of a first primer) to reduce thepercentage of beads that react with more than one polynucleotide. Insome embodiments, nucleic acid amplification reactions can be conductedwith a polynucleotide-to-bead ratio that is selected to optimize thepercentage of beads having a monoclonal population of polynucleotidesattached thereto. For example, a nucleic acid amplification reaction canbe conducted at anyone of polynucleotide-to-bead ratios in a range ofabout 1:2 to 1:500. In some embodiments, a polynucleotide-to-bead ratioincludes about 1:2, or about 1:5, or about 1:10, or about 1:25, or about1:50, or about 1:75, or about 1:100, or about 1:125, or about 1:150, orabout 1:175, or about 1:200, or about 1:225, or about 1:225, or about1:250. In some embodiments, a nucleic acid amplification reaction canproduce beads having zero types of polynucleotides attached thereto,other beads having one type of polynucleotide attached thereto, andother beads having more than one type of polynucleotides attachedthereto.

In some embodiments, the reaction mixture comprises one or more primers.For example, the reaction mixture can include at least a firstoligonucleotide primer. In some embodiments, a first primer can includea forward amplification primer which hybridizes to at least a portion ofone strand of a polynucleotide. In some embodiments, a first primercomprises an extendible 3′ end for nucleotide polymerization.

In some embodiments, methods for nucleic acid amplification comprisehybridization to the template of additional primers. For example, asecond primer can be a reverse amplification primer which hybridizes toat least a portion of one strand of a polynucleotide. In someembodiments, a second primer comprises an extendible 3′ end. In someembodiment, a second primer is not attached to a surface.

In some embodiments, a third primer can be a forward amplificationprimer which hybridizes to at least a portion of one strand of apolynucleotide. In some embodiments, a third primer comprises anextendible 3′ end. In some embodiment, a third primer is not attached toa surface. In some embodiments, a third primer comprises a bindingpartner or affinity moiety (e.g., biotin) for enriching the amplifiednucleic acids.

In some embodiments, primers (e.g., first, second and third primers)comprise single-stranded oligonucleotides.

In some embodiments, at least a portion of a primer can hybridize with aportion of at least one strand of a polynucleotide in the reactionmixture. For example, at least a portion of a primer can hybridize witha nucleic acid adaptor that is joined to one or both ends of thepolynucleotide. In some embodiments, at least a portion of a primer canbe partially or fully complementary to a portion of the polynucleotideor to the nucleic acid adaptor. In some embodiments, a primer can becompatible for use in any type of sequencing platform including chemicaldegradation, chain-termination, sequence-by-synthesis, pyrophosphate,massively parallel, ion-sensitive, and single molecule platforms.

In some embodiments, a primer (e.g., first, second or third primer) canhave a 5′ or 3′ overhang tail (tailed primer) that does not hybridizewith a portion of at least one strand of a polynucleotide in thereaction mixture. In some embodiments, a tailed primer can be anylength, including 1-50 or more nucleotides in length.

In some embodiments, primers comprise polymers of deoxyribonucleotides,ribonucleotides, and/or analogs thereof. In some embodiments, primerscomprise naturally-occurring, synthetic, recombinant, cloned, amplified,or unamplified forms. In some embodiments, primers comprise DNA, cDNARNA, chimeric RNA/DNA, or nucleic acid analogs.

In some embodiments, primers can be any length, including about 5-10nucleotides, or about 10-25 nucleotides, or about 25-40 nucleotides, orabout 40-55 nucleotides, or longer.

In some embodiments, methods for nucleic acid amplification can includeone or more different polymerases. In some embodiments, a polymeraseincludes any enzyme, or fragment or subunit of thereof, that cancatalyze polymerization of nucleotides and/or nucleotide analogs. Insome embodiments, a polymerase requires an extendible 3′ end. Forexample, a polymerase requires a terminal 3′ OH of a nucleic acid primerto initiate nucleotide polymerization.

A polymerase comprises any enzyme that can catalyze the polymerizationof nucleotides (including analogs thereof) into a nucleic acid strand.Typically but not necessarily such nucleotide polymerization can occurin a template-dependent fashion. In some embodiments, a polymerase canbe a high fidelity polymerase. Such polymerases can include withoutlimitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerase, or the linkageof parts of two or more polymerases. The term “polymerase” and itsvariants, as used herein, also refers to fusion proteins comprising atleast two portions linked to each other, where the first portioncomprises a peptide that can catalyze the polymerization of nucleotidesinto a nucleic acid strand and is linked to a second portion thatcomprises a second polypeptide, such as, for example, a reporter enzymeor a processivity-enhancing domain. Typically, the polymerase comprisesone or more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. In some embodiments, a polymeraseincludes or lacks other enzymatic activities, such as for example, 3′ to5′ exonuclease activity or 5′ to 3′ exonuclease activity. In someembodiments, a polymerase can be isolated from a cell, or generatedusing recombinant DNA technology or chemical synthesis methods. In someembodiments, a polymerase can be expressed in prokaryote, eukaryote,viral, or phage organisms. In some embodiments, a polymerase can bepost-translationally modified proteins or fragments thereof.

In some embodiments, the polymerase can include any one or morepolymerases, or biologically active fragment of a polymerase, which isdescribed in U.S. Patent Publ. No. 2011/0262903 to Davidson et al.,published Oct. 27, 2011, and/or International PCT Publ. No. WO2013/023176 to Vander Horn et al., published Feb. 14, 2013.

In some embodiments, a polymerase can be a DNA polymerase and includewithout limitation bacterial DNA polymerases, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases.

In some embodiments, a polymerase can be a replicase, DNA-dependentpolymerase, primases, RNA-dependent polymerase (including RNA-dependentDNA polymerases such as, for example, reverse transcriptases), athermo-labile polymerase, or a thermo-stable polymerase. In someembodiments, a polymerase can be any Family A or B type polymerase. Manytypes of Family A (e.g., E. coli Pol I), B (e.g., E. coli Pol II), C(e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g., humanPol beta), and Y (e.g., E. coli UmuC/DinB and eukaryotic RAD30/xerodermapigmentosum variants) polymerases are described in Rothwell and Watsman2005 Advances in Protein Chemistry 71:401-440. In some embodiments, apolymerase can be a T3, T5, T7, or SP6 RNA polymerase.

In some embodiments, nucleic acid amplification reactions can beconducted with one type or a mixture of polymerases, recombinases and/orligases. In some embodiments, nucleic acid amplification reactions canbe conducted with a low fidelity or high fidelity polymerase.

In some embodiments, nucleic acid amplification reactions can becatalyzed by heat-stable or heat-labile polymerases.

In some embodiment, a polymerase can lack 5′-3′ exonuclease activity. Insome embodiments, a polymerase can have strand-displacement activity.

In some embodiments, an archaeal DNA polymerase can be, withoutlimitation, a thermostable or thermophilic DNA polymerase such as, forexample: a Bacillus subtilis (Bsu) DNA polymerase I large fragment; aThermus aquaticus (Taq) DNA polymerase; a Thermus filiformis (Tfi) DNApolymerase; a Phi29 DNA polymerase; a Bacillus stearothermophilus (Bst)DNA polymerase; a Thermococcus sp. 9° N-7 DNA polymerase; a Bacillussmithii (Bsm) DNA polymerase large fragment; a Thermococcus litoralis(Tli) DNA polymerase or Vent™ (exo−) DNA polymerase (from New EnglandBiolabs); or “Deep Vent” (exo-) DNA polymerase (New England Biolabs).

In some embodiments, methods for nucleic acid amplification can includeone or more types of nucleotides. A nucleotide comprises any compoundthat can bind selectively to, or can be polymerized by, a polymerase.Typically, but not necessarily, selective binding of the nucleotide tothe polymerase is followed by polymerization of the nucleotide into anucleic acid strand by the polymerase; occasionally however thenucleotide may dissociate from the polymerase without becomingincorporated into the nucleic acid strand, an event referred to hereinas a “non-productive” event. Such nucleotides include not only naturallyoccurring nucleotides but also any analogs, regardless of theirstructure, that can bind selectively to, or can be polymerized by, apolymerase. While naturally occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the presentdisclosure can include compounds lacking any one, some or all of suchmoieties. In some embodiments, the nucleotide can optionally include achain of phosphorus atoms comprising three, four, five, six, seven,eight, nine, ten or more phosphorus atoms. In some embodiments, thephosphorus chain can be attached to any carbon of a sugar ring, such asthe 5′ carbon. The phosphorus chain can be linked to the sugar with anintervening O or S. In one embodiment, one or more phosphorus atoms inthe chain can be part of a phosphate group having P and O. In anotherembodiment, the phosphorus atoms in the chain can be linked togetherwith intervening O, NH, S, methylene, substituted methylene, ethylene,substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where Rcan be a 4-pyridine or 1-imidazole). In one embodiment, the phosphorusatoms in the chain can have side groups having O, BH₃, or S. In thephosphorus chain, a phosphorus atom with a side group other than O canbe a substituted phosphate group. In the phosphorus chain, phosphorusatoms with an intervening atom other than O can be a substitutedphosphate group. Some examples of nucleotide analogs are described inXu, U.S. Pat. No. 7,405,281.

Some examples of nucleotides that can be used in the disclosed methodsand compositions include, but are not limited to, ribonucleotides,deoxyribonucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotidepolyphosphates, modified ribonucleotide polyphosphates, modifieddeoxyribonucleotide polyphosphates, peptide nucleotides, modifiedpeptide nucleotides, metallonucleosides, phosphonate nucleosides, andmodified phosphate-sugar backbone nucleotides, analogs, derivatives, orvariants of the foregoing compounds, and the like. In some embodiments,the nucleotide can comprise non-oxygen moieties such as, for example,thio- or borano-moieties, in place of the oxygen moiety bridging thealpha phosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

In some embodiments, the nucleotide is unlabeled. In some embodiments,the nucleotide comprises a label and referred to herein as a “labelednucleotide”. In some embodiments, the label can be in the form of afluorescent dye attached to any portion of a nucleotide including abase, sugar or any intervening phosphate group or a terminal phosphategroup, i.e., the phosphate group most distal from the sugar.

In some embodiments, a nucleotide (or analog thereof) can be attached toa label. In some embodiments, the label comprises an opticallydetectable moiety. In some embodiments, the label comprises a moiety nottypically present in naturally occurring nucleotides. For example, thelabel can include fluorescent, luminescent or radioactive moieties.

In some embodiments, methods for nucleic acid amplification can furthercomprise an enrichment step. In some embodiments, methods for nucleicacid amplification can produce at least one bead attached with aplurality of polynucleotides (e.g., amplified nucleic acids) having asequence that is complementary to a template polynucleotide. At leastone of the polynucleotides attached to the bead can be hybridized to apolynucleotide having a biotinylated moiety (e.g., a reverseamplification product with the third primer). In some embodiments, anenrichment step comprises forming a purification complex by binding thepolynucleotide having a biotinylated moiety with a purification bead(e.g., paramagnetic bead) that is attached to a streptavidin moiety(e.g., (MyOne™ Bead from Dynabeads). In some embodiments, thepurification complex can be separated/removed from the reaction mixtureby attraction with a magnet.

In some embodiments, any combination or subcombination of reagents forconducting a nucleic acid amplification reaction can be contacted witheach other to form an amplification reaction mixture. For example, thereagents can be deposited into a reaction vessel, or can be delivered toan array of reaction chambers, in any order, including sequentially orsimultaneously (including substantially simultaneously), or acombination of sequentially for some reagents and simultaneously forother reagents. In some embodiments, reagents for conducting a nucleicacid amplification reaction include any one or more of the following: atleast one support (e.g., a bead or a surface of the site or reactionchamber), polynucleotides, recombinase, polymerase, oligonucleotideprimers (e.g., first, second, third, or additional oligonucleotideprimers), nucleotides, divalent cations, ATP, co-factors, sievingagents, diffusion reducing agents and accessory proteins (e.g.,helicase, single-stranded binding proteins and recombinase loadingfactor). Optionally, the nucleic acid amplification reaction can beconducted in a single continuous liquid phase or in an emulsion,including emulsions that provide compartmentalization and bicontinuousmicroemulsions.

In some embodiments, amplicons including substantially monoclonalnucleic acid populations are individually deposited, distributed orpositioned to different sites in an array of sites.

In some embodiments, the disclosed methods include distributing,depositing or otherwise positioning a single template molecule (e.g., asingle target polynucleotide of a sample) into a reaction chamber orsite (e.g., within an array). A single polynucleotide can be distributedfrom a sample into a reaction chamber by flowing a fluid having thesample of polynucleotides over the reaction chamber. A singlepolynucleotide distributed into a reaction chamber can besingle-stranded or double-stranded. In some embodiments, the nucleicacid is amplified in the reaction chamber or site after thedistributing.

In some embodiments, different single target polynucleotides can bedistributed from a sample into each of different reaction chambersarranged in an array. Different single polynucleotides can bedistributed from a sample into each of different reaction chambers byflowing a fluid having the sample of polynucleotides over the reactionchambers. Different single polynucleotides distributed into each ofdifferent reaction chambers can be single-stranded or double-stranded.

In some embodiments, the methods include distributing a singlepolynucleotide into a reaction chamber, and amplifying the singlepolynucleotide within the reaction chamber, thereby producing amonoclonal nucleic acid population in the reaction chamber.

In some embodiments, methods for distributing a single targetpolynucleotide into the reaction chamber and amplifying the singletarget polynucleotide comprise a nucleic acid sample. In someembodiments, a single polynucleotide, or different singlepolynucleotides, can be distributed from a nucleic acid sample having aplurality of polynucleotides. For example, a nucleic acid sample caninclude about 2-10, or about 10-50, or about 50-100, or about 100-500,or about 500-1,000, or about 1,000-5,000, or about 10³-10⁶, or moredifferent polynucleotides. Different polynucleotides can have the sameor different sequences. Different polynucleotides can have the same ordifferent lengths. A sample can include single-stranded ordouble-stranded polynucleotides or a mixture of both.

In some embodiments, methods for distributing a single targetpolynucleotide into the reaction chamber and amplifying the singletarget polynucleotide comprise distributing single polynucleotides. Insome embodiments, single polynucleotides can include single-stranded anddouble-stranded nucleic acid molecules. In some embodiments, nucleicacids can include polymers of deoxyribonucleotides, ribonucleotides,and/or analogs thereof. In some embodiments, nucleic acids can includenaturally-occurring, synthetic, recombinant, cloned, amplified,unamplified or archived (e.g., preserved) forms. In some embodiments,nucleic acids can include DNA, cDNA RNA or chimeric RNA/DNA, and nucleicacid analogs. In some embodiments, single polynucleotides can includenucleic acid library constructs comprising a nucleic acid joined at oneor both ends to an oligonucleotide adaptor. In some embodiments, nucleicacid library constructs can be compatible for use in any type ofsequencing platform including chemical degradation, chain-termination,sequence-by-synthesis, pyrophosphate, massively parallel, ion-sensitive,and single molecule platforms.

In some embodiments, the sites of an array can include one or morereaction chambers can be a well on a solid surface. A reaction chambercan have walls that define width and depth. The dimensions of a reactionchamber can be sufficient to permit deposition of reagents or forconducting reactions. A reaction chamber can have any shape includingcylindrical, polygonal or a combination of different shapes. Any wall ofa reaction chamber can have a smooth or irregular surface. A reactionchamber can have a bottom with a planar, concave or convex surface. Thebottom and side walls of a reaction chamber can comprise the same ordifferent material and/or can be coated with a chemical group that canreact with a biomolecule such as nucleic acids, proteins or enzymes.

In some embodiments, the reaction chamber can be one of multiplereaction chambers arranged in a grid or array. An array can include twoor more reaction chambers. Multiple reaction chambers can be arrangedrandomly or in an ordered array. An ordered array can include reactionchambers arranged in a row, or in a two-dimensional grid with rows andcolumns.

An array can include any number of reaction chambers for depositingreagents and conducting numerous individual reactions. For example, anarray can include at least 256 reaction chambers, or at least 256,000,or at least 1-3 million, or at least 3-5 million, or at least 5-7million, or at least 7-9 million, at least 9-11 million, at least 11-13million reaction chambers, or even high density including 13-700 millionreaction chambers or more. Reaction chambers arranged in a grid can havea center-to-center distance between adjacent reaction chambers (e.g.,pitch) of less than about 10 microns, or less than about 5 microns, orless than about 1 microns, or less than about 0.5 microns.

An array can include reaction chambers having any width and depthdimensions. For example, a reaction chamber can have dimensions toaccommodate a single microparticle (e.g., microbead) or multiplemicroparticles. A reaction chamber can hold 0.001-100 picoliters ofaqueous volume.

In some embodiments, at least one reaction chamber can be coupled to oneor more sensors or can be fabricated above one or more sensors. Areaction chamber that is coupled to a sensor can provide confinement ofreagents deposited therein so that products from a reaction can bedetected by the sensor. A sensor can detect changes in products from anytype of reaction, including any nucleic acid reaction such as primerextension, amplification or nucleotide incorporation reactions. A sensorcan detect changes in ions (e.g., hydrogen ions), protons, phosphategroups such as pyrophosphate groups. In some embodiments, at least onereaction chamber can be coupled to one or more ion sensitive fieldeffect transistor (ISFET). Examples of an array of reaction chamberscoupled to ISFET sensors can be found at U.S. Pat. No. 7,948,015, andU.S. Ser. No. 12/002,781.

In some embodiments, the amplification methods (as well as relatingcompositions, systems and apparatuses) described herein can be practicedin an array of reaction chambers, where the reaction chambers of thearray form part of a single fluidic system. In some embodiments, anarray of multiple reaction chambers can include a fluidic interfacewhich flows fluids (e.g., liquid or gas) across the reaction chambers ina controlled laminar flow. In some embodiments, an array of reactionchambers can include a fluid head space above the reaction chambers forlaminar flow. In some embodiments, an array of reaction chambers can bepart of a flow cell or flow chamber, where the reaction chambers are influid communication with each other. For example, a fluid can flow ontoan array to at least partially or fully fill one or multiple reactionchambers of the array. In some embodiments, a fluid can completely fillmultiple reaction chambers and the excess fluid can flood the top of thereaction chambers to form a fluid layer on top of the reaction chambers.The fluid layer on top of the reaction chambers can provide fluidcommunication with multiple reaction chambers in the array. In someembodiments, a fluid communication among multiple reaction chambers inan array can be used to conduct separate parallel reactions in themultiple reaction chambers. For example, fluid communication can be usedto deliver polynucleotides and/or reagents to multiple reaction chambersfor conducting parallel nucleic acid amplification reactions.

In some embodiments, sample having a plurality of differentpolynucleotides can be applied to a flow chamber to distribute singlepolynucleotides to individual reaction chambers in the array. In someembodiments, additional reagents can be applied to the flow chamber fordistribution into individual reaction chambers in the array. Forexample, additional reaction reagents can include microparticles, one ormore enzymes, enzyme co-factors, primers and/or nucleosidetriphosphates. In some embodiments, polynucleotides and reagents can bedelivered to an array of reaction chambers in any order, includingsequentially or substantially simultaneously or a combination of both.For example, in some embodiments, polynucleotides can be distributed toan array of reaction chambers first followed by reagents, or the reverseorder can be used, or polynucleotides and reagents can be distributedessentially simultaneously.

In some embodiments, any means (including flow chamber) can be used todeliver polynucleotides and/or reagents to a percentage of reactionchambers in an array. For example, the percentage of reaction chambersin an array that are loaded includes about 1-25%, or about 25%, or about30%, or about 40%, or about 50%, or about 60%, or about 70%, or higherpercentages. In some embodiments, the percentage of reaction chambersloaded with polynucleotides and/or reagents can be increased byconducting two or more rounds of loading steps. For example, (a) in afirst round polynucleotides and/or reagents can be distributed tomultiple reaction chambers in an array, and (b) in a second roundpolynucleotides and/or reagents can be distributed to the same array.Additional loading rounds (e.g., third, fourth or more round) can beconducted. In some embodiments, any type of reaction can be conductedbetween any of the loading rounds and/or any type of reaction can beconducted after multiple loading rounds are completed. For example, anucleic acid amplification reaction can be conducted between any of theloading rounds or a nucleic acid amplification reaction can be conductedafter multiple loading rounds are completed. In some embodiments, aftereach loading round, a compound can be layered on the array to preventmigration of the polynucleotides and beads out of the reaction chambers.For example, after each loading round, a solution containing at leastone sieving agent can be layered on the array. In some embodiments, thesieving agent comprises a cellulose derivative. Alternatively, an oillayer can be layered on top of the reaction mixture within wells orchambers of the array.

In some embodiments, the disclosed methods (and related compositions,systems and kits) further include attaching the nucleic acid template toa site of the array prior to amplification of the template. Optionally,the site includes a primer, and the attaching includes hybridizing theprimer to the primer binding site of the template. For example, the sitewithin the array can include at least one immobilized primer thatincludes a sequence complementary to at least a portion of the primerbinding site of the template that is distributed or deposited at thesite. The primer facilitates attachment of the template to the array. Insome embodiments, a substantial portion of the sites in the arrayinclude at least one primer. The primers of different sites can beidentical to each other. Alternatively, the primers of different sitescan be different from each other. In one exemplary embodiment, at leasttwo sites each include a different target-specific primer.

The primer may be attached to the site of the array using any suitablemeans. In order to attach primers to the surface of a nanoarray ofreaction chambers (for example, an ISFET array of the type using inion-based sequencing), it can be useful to first synthesize or fabricatea three-dimensional matrix within at least some reaction chambers of thearray. In an embodiment, polymer matrix precursors can be applied to anarray of wells associated with one or more sensors. The polymer matrixprecursors can be polymerized to form an array of polymer matrices.These polymer matrices can be conjugated to oligonucleotides and can beuseful in various analytical techniques including genetic sequencingtechniques.

In some embodiments, hydrophilic polymer matrices are distributed inwells associated with sensors, such as sensors of a sensor array. In anexample, the hydrophilic matrices are as hydrogel matrices. Thehydrophilic matrices can correspond with sensors of the sensor array ina one-to-one configuration. In a further example, the sensors of thesensor array can include field effect transistor (FET) sensors, such asion sensitive field effect transistors (ISFET). In particular, thematrix material is cured-in-place and conforms to the configuration ofindividual wells of a sensing device. The interstitial areas between thewells can be substantially free of the polymer matrix. In an example,the matrix material can be bonded, such as covalently bonded, to asurface of the wells. In an example, the wells have a depth or thicknessin a range of 100 nm to 10 micrometers. In another example, the wellscan have a characteristic diameter in a range of 0.1 micrometers to 2micrometers.

In an exemplary method, the polymer matrix array can be formed byapplying an aqueous solution including polymer precursors into wells ofa well array. Volumes of the aqueous material defined by the well arraycan be isolated using an immiscible fluid disposed over the well array.The isolated volumes of the solution can be initiated to facilitatepolymerization of the matrix precursors, resulting in a matrix arraydistributed within the wells. In an example, an aqueous solutionincluding matrix precursors is distributed to the wells of the sensingdevice by flowing the aqueous precursor over the wells. In anotherexample, the aqueous solution is included as a dispersed phase within anemulsion. The dispersed phase can settle or be motivated into wells ofthe well array. The polymerization of the matrix precursors can beinitiated using initiators disposed within the aqueous phase or withinthe immiscible fluid. In another example, the polymerization can beinitiated thermally.

In another exemplary method, a matrix array can be formed within wellsof a sensing device by anchoring initiator molecules to a surface withinwells of the well array. The solution including matrix precursors can beprovided over the wells of the well array. An initiator can initiatedpolymerization of the matrix precursors, resulting in the formation ofthe polymer matrix within wells of the well array. In a further example,aspects of the above methods can be combined to further enhanceformation of the matrix arrays.

In a particular embodiment, a sequencing system includes a flow cell inwhich a sensory array is disposed, includes communication circuitry inelectronic communication with the sensory array, and includes containersand fluid controls in fluidic communication with the flow cell. In anexample, FIG. 13 illustrates an expanded and cross-sectional view of aflow cell 100 and illustrates a portion of a flow chamber 106. A reagentflow 108 flows across a surface of a well array 102, in which thereagent flow 108 flows over the open ends of wells of the well array102. The well array 102 and a sensor array 105 together can form anintegrated unit forming a lower wall (or floor) of flow cell 100. Areference electrode 104 can be fluidly coupled to flow chamber 106.Further, a flow cell cover 130 encapsulates flow chamber 106 to containreagent flow 108 within a confined region.

FIG. 14 illustrates an expanded view of a well 201 and a sensor 214, asillustrated at 110 of FIG. 13 . The volume, shape, aspect ratio (such asbase width-to-well depth ratio), and other dimensional characteristicsof the wells can be selected based on the nature of the reaction takingplace, as well as the reagents, byproducts, or labeling techniques (ifany) that are employed. The sensor 214 can be a chemical field-effecttransistor (chemFET), more specifically an ion-sensitive FET (ISFET),with a floating gate 218 having a sensor plate 220 optionally separatedfrom the well interior by a material layer 216. In addition, aconductive layer (not illustrated) can be disposed over the sensor plate220. In an example, the material layer 216 includes an ion sensitivematerial layer. The material layer 216 can be a ceramic layer, such asan oxide of zirconium, hafnium, tantalum, aluminum, or titanium, amongothers, or a nitride of titanium. In an example, the material layer 216can have a thickness in a range of 5 nm to 100 nm, such as a range of 10nm to 70 nm, a range of 15 nm to 65 nm, or even a range of 20 nm to 50nm.

While the material layer 216 is illustrated as extending beyond thebounds of the illustrated FET component, the material layer 216 canextend along the bottom of the well 201 and optionally along the wallsof the well 201. The sensor 214 can be responsive to (and generate anoutput signal related to) the amount of a charge 224 present on materiallayer 216 opposite the sensor plate 220. Changes in the charge 224 cancause changes in a current between a source 221 and a drain 222 of thechemFET. In turn, the chemFET can be used directly to provide acurrent-based output signal or indirectly with additional circuitry toprovide a voltage-based output signal. Reactants, wash solutions, andother reagents can move in and out of the wells by a diffusion mechanism240.

In an embodiment, reactions carried out in the well 201 can beanalytical reactions to identify or determine characteristics orproperties of an analyte of interest. Such reactions can generatedirectly or indirectly byproducts that affect the amount of chargeadjacent to the sensor plate 220. If such byproducts are produced insmall amounts or rapidly decay or react with other constituents,multiple copies of the same analyte can be analyzed in the well 201 atthe same time in order to increase the output signal generated. In anembodiment, multiple copies of an analyte can be attached to a solidphase support 212, either before or after deposition into the well 201.The solid phase support 212 can be a polymer matrix, such as ahydrophilic polymer matrix, for example, a hydrogel matrix or the like.For simplicity and ease of explanation, solid phase support 212 is alsoreferred herein as a polymer matrix.

The well 201 can be defined by a wall structure, which can be formed ofone or more layers of material. In an example, the wall structure canhave a thickness extending from the lower surface to the upper surfaceof the well in a range of 0.01 micrometers to 10 micrometers, such as arange of 0.05 micrometers to 10 micrometers, a range of 0.1 micrometersto 10 micrometers, a range of 0.3 micrometers to 10 micrometers, or arange of 0.5 micrometers to 6 micrometers. In particular, the thicknesscan be in a range of 0.01 micrometers to 1 micrometer, such as a rangeof 0.05 micrometers to 0.5 micrometers, or a range of 0.05 micrometersto 0.3 micrometers. The wells 201 can have a characteristic diameter,defined as the square root of 4 times the cross-sectional area (A)divided by Pi (e.g., sqrt(4*A/π), of not greater than 5 micrometers,such as not greater than 3.5 micrometers, not greater than 2.0micrometers, not greater than 1.6 micrometers, not greater than 1.0micrometers, not greater than 0.8 micrometers or even not greater than0.6 micrometers. In an example, the wells 201 can have a characteristicdiameter of at least 0.01 micrometers.

While FIG. 14 illustrates a single-layer wall structure and asingle-layer material layer 216, the system can include, one or morewall structure layers, one or more conductive layers or one or morematerial layers. For example, the wall structure can be formed of one ormore layers, including an oxide of silicon or TEOS or including anitride of silicon.

In a particular example illustrated in FIG. 15 , a system 300 includes awell wall structure 302 defining an array of wells 304 disposed over oroperatively coupled to sensor pads of a sensor array. The well wallstructure 302 defines an upper surface 306. A lower surface 308associated with the well is disposed over a sensor pad of the sensorarray. The well wall structure 302 defines a sidewall 310 between theupper surface 306 and the lower surface 308. As described above, amaterial layer in contact with sensor pads of the sensor array canextend along the lower surface 308 of a well of the array of wells 304or along at least a portion of the wall 310 defined by the well wallstructure 302. The upper surface 306 can be free of the material layer.In particular, a polymer matrix can be disposed in the wells of thearray of wells 304. The upper surface 306 can be substantially free ofthe polymer matrix. For example, the upper surface 306 can include anarea that is free of the polymer matrix, such as at least 70% of thetotal area, at least 80% of the total area, at least 90% of the totalarea or approximately 100% of the total area.

While the wall surface of FIG. 14 is illustrated as extendingsubstantially vertically and outwardly, the wall surface can extend invarious directions and have various shapes. Substantially verticallydenotes extending in a direction having a component that is normal tothe surface defined by the sensor pad. For example, as illustrated inFIG. 16 , a well wall 402 can extend vertically, being parallel to anormal component 412 of a surface defined by a sensor pad. In anotherexample, the wall surface 404 extends substantially vertically, in anoutward direction away from the sensor pad, providing a larger openingto the well than the area of the lower surface of the well. Asillustrated in FIG. 16 , the wall surface 404 extends in a directionhaving a vertical component parallel to the normal component 412 of thesurface 414. In an alternative example, a wall surface 406 extendssubstantially vertically in an inward direction, providing an openingarea that is smaller than an area of the lower surface of the well. Thewall surface 406 extends in a direction having a component parallel tothe normal component 412 of the surface 414.

While the surfaces 402, 404, or 406 are illustrated by straight lines,some semiconductor or CMOS manufacturing processes can result instructures having nonlinear shapes. In particular, wall surfaces, suchas wall surface 408 and upper surfaces, such as upper surface 410, canbe arcuate in shape or take various nonlinear forms. While thestructures and devices illustrated herewith are depicted as havinglinear layers, surfaces, or shapes, actual layers, surfaces, or shapesresulting from semiconductor processing can differ to some degree,possibly including nonlinear and arcuate variations of the illustratedembodiment.

FIG. 17 includes an illustration of exemplary wells including ionsensitive material layers. For example, a well structure 502 can definean array of wells, such as exemplary wells 504, 506, or 508. The wells(504, 506, or 508) can be operatively coupled to an underlying sensor(not illustrated) or linked to such an underlying sensor. Exemplary well504 includes an ion sensitive material layer 510 defining the bottom ofthe well 504 and extending into the structure 502. While not illustratedin FIG. 17 , a conductive layer, such as a gate, for example, a floatinggate of ion sensitive field effect transistor can reside below the ionsensitive material layer 510.

In another example, as illustrated by well 506, an ion sensitivematerial layer 512 can define the bottom of the well 506 withoutextending into the structure 502. In a further example, a well 508 caninclude an ion sensitive layer 514 that extends along at least a portionof a sidewall 516 of the well 508 defined by the structure 502. Asabove, the ion sensitive material layers 512 or 514 can reside overconductive layers or gates of underlying electronic devices.

Returning to FIG. 14 , the matrix material 212 is conformal with thewell structure. In particular, the matrix material can be cured in placeto be conformal to the walls and bottom surface of the well. An uppersurface through which the wells are defined can include an area that issubstantially free of the matrix material, such as at least 70% of thetotal area, at least 80% of the total area, at least 90% of the totalarea or approximately 100% of the total area. Depending upon the natureof the well structure, the polymer matrix can be physically secured tothe well wall structure. In another example, the polymer matrix can bechemically bound to the well wall structure. In particular, the polymermatrix can be covalently bound to the well wall structure. In anotherexample, the polymer matrix can be bound by hydrogen bonding or ionicbonding to the well wall structure.

The polymer matrix can be formed from matrix precursors, such as aradically polymerizable monomer, such as a vinyl-based monomer. Inparticular, the monomer can include a hydrophilic monomer, such as anacrylamide, vinyl acetate, hydroxyalkylmethacrylate, variations orderivatives thereof, copolymers thereof, or any combination thereof. Ina particular example, the hydrophilic monomer is an acrylamide, such asan acrylamide functionalized to include hydroxyl groups, amino groups,carboxyl groups, halogen groups, or a combination thereof. In anexample, the hydrophilic monomer is an aminoalkyl acrylamide, anacrylamide functionalized with an amine terminated polypropylene glycol(D, illustrated below), an acrylopiperazine (C, illustrated below), or acombination thereof. In another example, the acrylamide can be ahydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. In particular,the hydroxyalkyl acrylamide can includeN-tris(hydroxymethyl)methyl)acrylamide (A, illustrated below),N-(hydroxymethyl)acrylamide (B, illustrated below), or a combinationthereof. In another example, the a comonomer can include a halogenmodified acrylate or acrylamide, such as aN-(5-bromoacetamidylpentyl)acrylamide (BRAPA, E, illustrated below). Inanother example, a comonomer can include an oligonucleotide modifiedacrylate or acrylamide monomer. In a further example, a mixture ofmonomers, such as a mixture of hydroxyalky acrylamide and aminefunctionalize acrylamide or a mixture of acrylamide and aminefunctionalized acrylamide, can be used. In an example, the aminefunctionalize acrylamide can be included in a ratio of hydroxyalkylacrylamide:amine functionalized acrylamide or acrylamide:aminefunctionalized acrylamide in a range of 100:1 to 1:1, such as a range of100:1 to 2:1, a range of 50:1 to 3:1, a range of 50:1 to 5:1 or even arange of 50:1 to 10:1. In another example, the amine functionalizeacrylamide can be included in a ratio of hydroxyalkyl acrylamide:brominefunctionalized acrylamide or acrylamide:bromine functionalizedacrylamide in a range of 100:1 to 1:1, such as a range of 100:1 to 2:1,a range of 50:1 to 3:1, a range of 50:1 to 5:1 or even a range of 50:1to 10:1.

In a further example, an oligonucleotide functionalized acrylamide oracrylate monomer, such as an Acrydite™ monomer, can be included toincorporate oligonucleotides into the polymer matrix.

Another exemplary matrix precursor includes a crosslinker. In anexample, the crosslinker is included in a mass ratio of monomer tocrosslinker in a range of 15:1 to 1:2, such as a range of 10:1 to 1:1, arange of 6:1 to 1:1, or even a range of 4:1 to 1:1. In particular, thecrosslinker can be a divinyl crosslinker. For example, a divinylcrosslinker can include a diacrylamide, such asN,N′-(ethane-1,2-diyl)bis(2-hydroxyl ethyl)acrylamide,N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, or a combination thereof.In another example, a divinyl crosslinker includes ethyleneglycoldimethacrylate, divinylbenzene, hexamethylene bisacrylamide,trimethylolpropane trimethacrylate, a protected derivative thereof, or acombination thereof.

In one aspect, polymer networks comprise polyacrylamide gels with totalmonomer percentages in the range of from 3-20 percent, and morepreferably, in the range of from 5 to 10 percent. In one embodiment,crosslinker percentage of monomers is in the range of from 5 to 10percent. In a particular embodiment, polymer networks comprise 10percent total acrylamide of which 10 percent is bisacrylamidecrosslinker.

Polymerization can be initiated by an initiator within the solution. Forexample, the initiator can be a water-based. In another example, theinitiator can be a hydrophobic initiator, preferentially residing in ahydrophobic phase. An exemplary initiator includes ammonium persulfateor TEMED (tetramethylethylenediamine). TEMED can accelerate the rate offormation of free radicals from persulfate, in turn catalyzingpolymerization. The persulfate free radicals, for example, convertacrylamide monomers to free radicals which react with unactivatedmonomers to begin the polymerization chain reaction. The elongatingpolymer chains can be randomly crosslinked, resulting in a gel with acharacteristic porosity which depends on the polymerization conditionsand monomer concentrations. Riboflavin (or riboflavin-5′-phosphate) canalso be used as a source of free radicals, often in combination withTEMED and ammonium persulfate. In the presence of light and oxygen,riboflavin is converted to its leuco form, which is active in initiatingpolymerization, which is usually referred to as photochemicalpolymerization.

In another example, an azo initiator can be used to initiatepolymerization. Exemplary water soluble azo initiators are illustratedin Table 1 and exemplary oil soluble azo initiators are illustrated inTable 2. In particular, the azo initiator can be azobisisobutyronitrile(AIBN).

TABLE I Water Soluble Azo Initiator Compounds 10 hour half-lifedecomposition Structure temperature

44° C.

47° C.

56° C.

57° C.

60° C.

61° C.

67° C.

80° C.

87° C.

TABLE II Oil Soluble Azo Initiator Compounds 10 hour half-lifedecomposition Structure temperature

 30° C.

 51° C.

 66° C.

 67° C.

 88° C.

 96° C.

104° C.

110° C

111° C.

In a further example, precursors to the polymer matrix can includesurface reactive additives to enhance binding with surface. Exemplaryadditives include functionalize acrylic monomers or functionalizedacrylamide monomers. For example, an acrylic monomer can befunctionalized to bind with a surface material, such as a ceramicmaterial forming the bottom or sidewall of a well. In an example, theadditive can include an acryl-phosphonate, such as methacrylphosphonate.In another example, the additive can include dimethylacrylamide orpolydimethylacrylamide. In a further example, the additive can include apolylysine modified with polymerizable groups, such as acrylate groups.

In another example, polymerization can be facilitated using an atomtransfer radical polymerization (ATRP). The ATRP system can include achain transfer agent (CTA), monomer, a transition metal ion, and aligand. An exemplary transition metal ion complex includes acopper-based complex. An exemplary ligand includes 2,2′-bipyridine,4,4′-di-5-nonyl-2,2′-bipyridine,4,4′,4″-tris(5-nonyl)-2,2′:6′,2″-terpyridine,N,N,N′,N′,N″-pentamethyldiethylenetriamine,1,1,4,7,10,10-hexamethyltriethylenetetramine,tris(2-dimethylaminoethyl)amine, N,N-bis(2-pyridylmethyl)octadecylamine,N,N,N′,N′-tetra[(2-pyridyl)methyl]ethylenediamine,tris[(2-pyridyl)methyl]amine, tris(2-aminoethyl)amine,tris(2-bis(3-butoxy-3-oxopropyl)aminoethyl)amine,tris(2-bis(3-(2-ethylhexoxy)-3-oxopropyl)aminoethyl)amine,tris(2-bis(3-dodecoxy-3-oxopropyl)aminoethyl)amine, aliphatic, aromaticand heterocyclic/heteroaromatic amines, variations and derivativesthereof, or combinations thereof. An exemplary CTA includes2-bromopropanitrile, ethyl 2-bromoisobutyrate, ethyl 2-bromopropionate,methyl 2-bromopropionate, 1-phenyl ethylbromide, tosyl chloride,1-cyano-1-methylethyldiethyldithiocarbamate,2-(N,N-diethyldithiocarbamyl)-isobutyric acid ethyl ester, dimethyl2,6-dibromoheptanedioate, and other functionalized alkyl halides,variations or derivatives thereof, or any combination thereof.Optionally, the BRAPA monomer can function as a branching agent in thepresence of an ATRP system.

In an example, ATRP is initiated at a surface to directly bond thepolymer to the surface. For example, acrylate monomers, acrylamidemonomers, Acrydite™ monomers, succinimidyl acrylates, bis-acrylate orbis-acrylamide monomers, derivatives thereof, or combinations thereofcan be applied in solution to the initiated surface in the presence of atransition metal ion/ligand complex.

In another, the ATRP system can be used to attach a polymer to a surfaceof the well using a modified phosphonate, sulfonate, silicate, orzirconate compounds. In particular, an amine or hydroxyl terminatedalkyl phosphonate or an alkoxy derivative thereof can be applied to asurface and initiated using an initiator. The catalyst complex andmonomers can be applied, extending the surface compound.

In an exemplary method, an aqueous solution including precursors to thepolymer matrix can be applied into wells of the structure defining anarray of wells. The aqueous solution in the wells can be isolated byproviding an immiscible fluid over the wells and initiatingpolymerization of the polymer precursors within the solution within thewells.

For example, FIG. 18 illustrates an exemplary well structure 602defining wells 604. One or more sensors (not illustrated) can beoperatively coupled or linked to the wells 604. For example, one or moresensors can include gate structures in electrical communication to atleast the bottom surface of the wells 604. An aqueous solution 606 thatincludes polymer precursors, among other components, is provided overthe wells and distributes the solution including the polymer precursorsinto the wells 604. Exemplary polymer precursors include monomers,crosslinkers, initiators, or surface reactive agents, among others, suchas described above. Optionally, the wells 604 can be wet prior todeposition using a hydrophilic solution, such as a solution includingwater, alcohol, or mixtures thereof, or a solution including water and asurfactant. An exemplary alcohol includes isopropyl alcohol. In anotherexample, the alcohol includes ethanol. While not illustrated, the bottomsurface of the well and optionally the sidewall of the well can includean ion sensitive material. Such ion sensitive material can overlieconductive structure of an underlying electronic device, such as fieldeffect transistor. One or more surfaces of the well can be treated witha surface reactive additive prior to applying a solution includingpolymer precursors.

Distributing the aqueous solutions including polymer precursors into thewell 604 can be further enhanced by agitating the structure such asthrough spinning or vortexing. In another example, vibration, such assonic or supersonic vibrations, can be used to improve distribution ofthe aqueous solution within the wells 604. In a further example, thewells can be degassed using a vacuum and the solutions applied whileunder a negative gauge pressure. In an example, the aqueous solution isdistributed to wells at room temperature. In another example, theaqueous solution is distributed at a temperature below room temperature,particularly when an aqueous-based initiator is used. Alternatively, theaqueous solution is distributed at an elevated temperature.

As illustrated in FIG. 19 , an immiscible fluid 708 is applied over thewell 604 pushing the aqueous solution 606 away from the top of the wellsand isolating the aqueous solution 606 within the wells 604, asillustrated in FIG. 20 . An exemplary immiscible fluid includes mineraloil, silicone oil (e.g., poly(dimethylsiloxane)), heptane, carbonateoils (e.g. diethylhexyl carbonate (Tegosoft DEC®), or combinationsthereof.

Initiators can be applied within the aqueous solution 606.Alternatively, initiators can be provided within the immiscible fluid708. Polymerization can be initiated by changing the temperature of thesubstrate. Alternatively, polymerization can occur at room temperature.In particular, the polymer precursor solution can be held at atemperature in a range of 20° C. to 100° C., such as a range of 25° C.to 90° C., a range of 25° C. to 50° C., or a range of 25° C. to 45° C.,for a period of time in a range of 10 minutes to 5 hours, such as arange of 10 minutes to 2 hours, or a range of 10 minutes to 1 hour.

As a result of the polymerization, an array of polymer matrices 912 areformed within the wells 604 defined by the well structure 602, asillustrated in FIG. 21 . Optionally, the array can be washed with NaOH(e.g., 1 N NaOH) to remove polymer from interstitial areas betweenwells.

In an alternative example, an emulsion including an aqueous solution ofpolymer precursors as a dispersed phase within an immiscible fluid canbe used to deposit droplets of aqueous solution within wells. Forexample, as illustrated in FIG. 22 , a well structure 1002 defines wells1004. The wells can be operatively coupled or electrically linked to oneor more sensors (not illustrated). As described above, the bottomsurface and optionally side surfaces of the walls of the wells 1004 canbe defined by ion sensitive material layers which can overlie conductivefeatures of underlying electronic devices.

An emulsion 1006 can include dispersed aqueous droplets 1008 thatinclude polymer precursors within a continuous immiscible fluid 1010.The droplets 1008 can settle into the wells 1004. In particular, theaqueous droplets 1008 have a density greater than the immiscible fluid1010. An exemplary immiscible fluid includes mineral oil, silicone oil(e.g., poly(dimethylsiloxane)) heptanes, carbonate oils (e.g.diethylhexyl carbonate (Tegosoft DEC®), or combinations thereof. In afurther example, distribution of the aqueous droplets into wells 1004can be facilitated by spinning, vortexing, or sonicating the fluid orstructures. Optionally, the wells 604 can be wet prior to depositionusing a hydrophilic solution, such as a solution including water,alcohol, or mixtures thereof, or a solution including water and asurfactant. The temperature during distribution of the droplets intowells can be performed at room temperature. Alternatively, thedistribution can be performed at an elevated temperature.

As illustrated in FIG. 23 , the droplets coalesce within the wells 1004to provide isolated solutions including polymer precursors 1112.Optionally, the emulsion 1006 can be replaced with an immiscible fluid,such as the immiscible fluid 1010 without droplets 1008 or a differentimmiscible fluid 1116. An exemplary immiscible fluid includes mineraloil, silicone oil (e.g., poly(dimethylsiloxane)) heptanes, carbonateoils (e.g. diethylhexyl carbonate (Tegosoft DEC®), or combinationsthereof. Alternatively, the emulsion 1006 can remain in place duringpolymerization. As such, the solutions 1112 within the wells 1004 areisolated from the solution in other wells 1004. Polymerization can beinitiated resulting in a polymer matrix 1214 within the wells 1004, asillustrated at FIG. 24 . As above, polymerization can be initiatedthermally. In another example, polymerization can be initiated using anoil phase initiator. Alternatively, polymerization can be initiatedusing an aqueous phase initiator. In particular, a second emulsion canbe applied over the wells 1004. The second emulsion can include adispersed aqueous phase including aqueous phase initiator.

Polymerization can be initiated by changing the temperature of thesubstrate. Alternatively, polymerization can occur at room temperature.In particular, the polymer precursor solution can be held at atemperature in a range of 20° C. to 100° C., such as a range of 25° C.to 90° C., a range of 25° C. to 50° C., or a range of 25° C. to 45° C.,for a period of time in a range of 10 minutes to 5 hours, such as arange of 10 minutes to 2 hours, or a range of 10 minutes to 1 hour.Optionally, the array can be washed with NaOH (e.g., 1 N NaOH) to removepolymer from interstitial areas between wells.

In another example, an array of matrix material can be formed withinwells of a well array using initiators secured to the surface of thewell array. For example, as illustrated in FIG. 25 , a structure 1302can define wells 1304. A material layer 1306 can define a lower surfaceof the wells 1304. Anchoring compounds 1308, such as anchoring compoundsuseful in Atom Transfer Radical Polymerization (ATRP), can be secured tothe material layer 1306 defining a bottom surface of the wells 1304.Alternatively, a sidewall material defined within the wells or layers ofthe structure 1302 expose within the wells 1304 can anchor compoundssuch as compounds useful in ATRP, as described above.

In such an example, a solution 1310 including polymer precursors such asmonomers, crosslinkers, and optionally surface reactive additive, can beapplied over the structure 1302 and within the wells 1304. The anchoringcompounds 1308 can be initiated to facilitate polymerization extendingfrom the anchoring compound, isolating the polymerization within thewells 1304 and securing the polymer to the well 1304. In an example, theanchoring compound has a surface reactive group and a distalradical-forming group. The surface reactive group can include aphosphonate, a silicate, a sulfonate, a zirconate, titanate or acombination thereof. The distal radical-forming group can include anamine or hydroxyl that can undergo transfer, for example, with ahalogenated (e.g., bromilated) compound and subsequently form afree-radical for use in polymerizing the polymer precursors andanchoring the resulting polymer to a well surface. In another example,the anchoring compound 1308 can include an alkyl bromoacetate modifiedwith a surface reactive group. For example, the anchoring compound 1308can include an alkylphosphono bromoacetate compound. The alkyl group caninclude between 3 and 16 carbons, such as between 6 and 16 carbons, orbetween 9 and 13 carbons. The alkyl group can be linear or branched. Inparticular, the alkyl group is linear. In a further example, thebromoacetyl group can be modified to include an alkyl modifiedbromoacetyl group, such as an ethyl or methyl modified bromoacetyl groupforming an ester with a surface functional alkyl group. In a particularexample, the anchoring compound includes the following compound:

Typically, an ATRP system can be selected to terminate polymerizationafter a statistical average length or number of monomer additions. Insuch a manner, the amount polymerization within a well 1304 can becontrolled. In a further example, other agents influencing chainextension or termination can be applied and added to the aqueoussolution 1310.

In an alternative example, an anchoring compound, such as describedabove, can be included in the polymer precursor solution, such asdescribed in relation to FIG. 18 or FIG. 22 . Following curing theanchoring compound can assist with bonding a polymer to the innersurfaces of a well.

In another example illustrated in FIG. 26 , a structure 1402 can definewells 1404. The wells 1404 can include a material layer 1406 thatextends along sidewalls 1410 of the structure 1402 and wells 1404. Theinitiator 1408 can be secured to the material layer 1406 along thebottom of the well 1404 and along the sidewalls 1410. A solution 1412including polymer precursors, crosslinkers, and other agents can beapplied over the structure 1402 and the wells 1404.

As illustrated in FIG. 27 , polymer matrices 1512 are formed as a resultof the initiated polymerization extending from surfaces within the wells1304, such as surfaces defined by the material layer 1306.

Polymerization can be initiated by changing the temperature of thesubstrate. Alternatively, polymerization can occur at room temperature.In particular, the polymer precursor solution can be held at atemperature in a range of 20° C. to 100° C., such as a range of 25° C.to 90° C., a range of 25° C. to 50° C., or a range of 25° C. to 45° C.,for a period of time in a range of 10 minutes to 5 hours, such as arange of 10 minutes to 2 hours, or a range of 10 minutes to 1 hour.

Once formed, the polymer matrices can be activated to facilitateconjugation with a target analyte, such as a polynucleotide. Forexample, functional groups on the polymer matrices can be enhanced topermit binding with target analytes or analyte receptors (e.g.,oligonucleotide primers). In a particular example, functional groups ofthe hydrophilic polymer matrix can be modified with reagents capable ofconverting the hydrophilic polymer functional groups to reactivemoieties that can undergo nucleophilic or electrophilic substitution.For example, hydroxyl groups on the polymer matrices can be activated byreplacing at least a portion of the hydroxyl groups with a sulfonategroup or chlorine. Exemplary sulfonate groups can be derived fromtresyl, mesyl, tosyl, or fosyl chloride, or any combination thereof.Sulfonate can act to permit nucleophiles to replace the sulfonate. Thesulfonate can further react with liberated chlorine to providechlorinated functional groups that can be used in a process to conjugatethe matrix. In another example, amine groups on the polymer matrices canbe activated.

For example, target analyte or analyte receptors can bind to thehydrophilic polymer through nucleophilic substitution with the sulfonategroup. In particular example, target analyte receptors terminated with anucleophile, such as an amine or a thiol, can undergo nucleophilicsubstitution to replace the sulfonate groups in the polymer matrices. Asa result of the activation, conjugated polymer matrices can be formed.

In another example, the sulfonated polymer matrices can be furtherreacted with mono- or multi-functional mono- or multi-nucleophilicreagents that can form an attachment to the matrix while maintainingnucleophilic activity for oligonucleotides comprising electrophilicgroups, such as maleimide. In addition, the residual nucleophilicactivity can be converted to electrophilic activity by attachment toreagents comprising multi-electrophilic groups, which are subsequentlyto attach to oligonucleotides comprising nucleophilic groups.

In another example, a monomer containing the functional group can beadded during the polymerization. The monomer can include, for example,an acrylamide containing a carboxylic acid, ester, halogen or otheramine reactive group. The ester group can be hydrolyzed before thereaction with an amine oligo.

Other conjugation techniques include the use of monomers that compriseamines. The amine group is a nucleophilic group that can be furthermodified with amine reactive bi-functional bis-electrophilic reagentsthat yield a mono-functional electrophilic group subsequent toattachment to the polymer matrix. Such an electrophilic group can bereacted with oligonucleotides having a nucleophilic group, such as anamine or thiol, causing attachment of the oligonucleotide by reactionwith the vacant electrophile.

If the polymer matrix is prepared from a combination of amino- andhydroxyl-acrylamides, the polymer matrix includes a combination ofnucleophilic amino groups and neutral hydroxyl groups. The amino groupscan be modified with di-functional bis-electrophilic moieties, such as adi-isocyanate or bis-NHS ester, resulting in a hydrophilic polymermatrix reactive to nucleophiles. An exemplary bis-NHS ester includesbis-succinimidyl C2-C12 alkyl esters, such as bis-succinimidyl suberateor bis-succinimidyl glutarate.

Other activation chemistries include incorporating multiple steps toconvert a specified functional group to accommodate specific desiredlinkages. For example, the sulfonate modified hydroxyl group can beconverted into a nucleophilic group through several methods. In anexample, reaction of the sulfonate with azide anion yields an azidesubstituted hydrophilic polymer. The azide can be used directly toconjugate to an acetylene substituted biomolecule via “CLICK” chemistrythat can be performed with or without copper catalysis. Optionally, theazide can be converted to amine by, for example, catalytic reductionwith hydrogen or reduction with an organic phosphine. The resultingamine can then be converted to an electrophilic group with a variety ofreagents, such as di-isocyanates, bis-NHS esters, cyanuric chloride, ora combination thereof. In an example, using di-isocyanates yields a urealinkage between the polymer and a linker that results in a residualisocyanate group that is capable of reacting with an amino substitutedbiomolecule to yield a urea linkage between the linker and thebiomolecule. In another example, using bis-NHS esters yields an amidelinkage between the polymer and the linker and a residual NHS estergroup that is capable of reacting with an amino substituted biomoleculeto yield an amide linkage between the linker and the biomolecule. In afurther example, using cyanuric chloride yields an amino-triazinelinkage between the polymer and the linker and two residualchloro-triazine groups one of which is capable of reacting with an aminosubstituted biomolecule to yield an amino-triazine linkage between thelinker and the biomolecule. Other nucleophilic groups can beincorporated into the matrix via sulfonate activation. For example,reaction of sulfonated matrices with thiobenzoic acid anion andhydrolysis of the consequent thiobenzoate incorporates a thiol into thematrix which can be subsequently reacted with a maleimide substitutedbiomolecule to yield a thio-succinimide linkage to the biomolecule.

Thiol can also be reacted with a bromo-acetyl group or bromo-amidylgroup. In a particular example, whenn-(5-bromoacetamidylpentyl)acrylamide (BRAPA) is included as acomonomer, oligonucleotides can be incorporated by forming athiobenzamide-oligonucleotide compound, for example, as illustratedbelow, for reacting with bromo-acetyl groups on the polymer.

The thiobenzamide-oligonucleotide compound can be formed by reacting thefollowing dithiobenzoate-NHS compound with an amine terminatedoligonucleotide and activating the dithiobenzamide-oligonucleotidecompound to form the above illustrated thiobenzamide-oligonucleotidecompound.

Alternatively, acrydite oligonucleotides can be used during thepolymerization to incorporate oligonucleotides. An exemplary acryditeoligonucleotide can include an ion-exchanged oligonucleotide.

Covalent linkages of biomolecules onto refractory or polymericsubstrates can be created using electrophilic moieties on the substratecoupled with nucleophilic moieties on the biomolecule or nucleophiliclinkages on the substrate coupled with electrophilic linkages on thebiomolecule. Because of the hydrophilic nature of most commonbiomolecules of interest, the solvent of choice for these couplings iswater or water containing some water soluble organic solvent in order todisperse the biomolecule onto the substrate. In particular,polynucleotides are generally coupled to substrates in water systemsbecause of their poly-anionic nature. Because water competes with thenucleophile for the electrophile by hydrolyzing the electrophile to aninactive moiety for conjugation, aqueous systems can result in lowyields of coupled product, where the yield is based on the electrophilicportion of the couple. When high yields of electrophilic portion of thereaction couple are desired, high concentrations of the nucleophiledrive the reaction and mitigate hydrolysis, resulting in inefficient useof the nucleophile. In the case of polynucleic acids, the metal counterion of the phosphate can be replaced with a lipophilic counter-ion, inorder to help solubilize the biomolecule in polar, non-reactive,non-aqueous solvents. These solvents can include amides or ureas such asformamide, N,N-dimethylformamide, acetamide, N,N-dimethylacetamide,hexamethylphosphoramide, pyrrolidone, N-methylpyrrolidone,N,N,N′,N′-tetramethylurea, N,N′-dimethyl-N,N′-trimethyleneurea, or acombination thereof; carbonates such as dimethyl carbonate, propylenecarbonate, or a combination thereof; ethers such as tetrahydrofuran;sulfoxides and sulfones such as dimethylsulfoxide, dimethylsulfone, or acombination thereof; hindered alcohols such as tert-butyl alcohol; or acombination thereof. Lipophilic cations can include tetraalkylammomiunor tetraarylammonium cations such as tetramethylamonium,tetraethylamonium, tetrapropylamonium, tetrabutylamonium,tetrapentylamonium, tetrahexylamonium, tetraheptylamonium,tetraoctylamonium, and alkyl and aryl mixtures thereof,tetraarylphosphonium cations such as tetraphenylphosphonium,tetraalkylarsonium or tetraarylarsonium such as tetraphenylarsonium, andtrialkylsulfonium cations such as trimethylsulfonium, or a combinationthereof. The conversion of polynucleic acids into organic solventsoluble materials by exchanging metal cations with lipophilic cationscan be performed by a variety of standard cation exchange techniques.

In a particular embodiment, the polymer matrices are exposed to targetpolynucleotides having a segment complementary to oligonucleotidesconjugated to the polymer matrices. The polynucleotides are subjected toamplification, such as through polymerase chain reaction (PCR) orrecombinase polymerase amplification (RPA). For example, the targetpolynucleotides are provided in low concentrations such that a singlepolynucleotide is likely to reside within a single polymer matrix of thearray of polymer matrices. The polymer matrix can be exposed to enzymes,nucleotides, salts or other components sufficient to facilitateduplication of the target polynucleotide.

In a particular embodiment, an enzyme such as a polymerase is present,bound to, or is in close proximity to the polymer matrix. A variety ofnucleic acid polymerase can be used in the methods described herein. Inan exemplary embodiment, the polymerase can include an enzyme, fragmentor subunit thereof, which can catalyze duplication of thepolynucleotide. In another embodiment, the polymerase can be anaturally-occurring polymerase, recombinant polymerase, mutantpolymerase, variant polymerase, fusion or otherwise engineeredpolymerase, chemically modified polymerase, synthetic molecules, oranalog, derivative or fragment thereof.

In some embodiments, methods for distributing a single targetpolynucleotide into the reaction chamber and amplifying the singletarget polynucleotide comprise a nucleic acid amplification reaction. Insome embodiments, any type of nucleic acid amplification reaction can beconducted including polymerase chain reaction (PCR) (U.S. Pat. Nos.4,683,195 and 4,683,202 both granted to Mullis), ligase chain reaction(LCR) (Barany 1991 Proceedings National Academy of Science USA88:189-193; Barnes 1994 Proceedings National Academy of ScienceUSA91:2216-2220), helicase-dependent amplification (HDA) or isothermalself-sustained sequence reaction (Kwoh 1989 Proceedings National Academyof Science USA 86:1173-1177; WO 1988/10315; and U.S. Pat. Nos.5,409,818, 5,399,491, and 5,194,370).

In some embodiments, the amplification reaction includes recombinasepolymerase amplification (RPA). (See, e.g., U.S. Pat. No. 5,223,414 toZarling, U.S. Pat. Nos. 5,273,881 and 5,670,316 both to Sena, and U.S.Pat. Nos. 7,270,981, 7,399,590, 7,435,561, 7,666,598, 7,763,427,8,017,339, 8,030,000, 8,062,850, and 8,071,308).

In some embodiments, methods for distributing a single targetpolynucleotide into the reaction chamber and amplifying the singletarget polynucleotide comprise an isothermal amplification condition. Insome embodiments, a nucleic acid amplification reaction can be conductedunder isothermal conditions. In some embodiments isothermalamplification conditions comprise a nucleic acid amplification reactionsubjected to a temperature variation which is constrained within alimited range during at least some portion of the amplification,including for example a temperature variation is within about 20° C., orabout 10° C., or about 5° C., or about 1-5° C., or about 0.1-1° C., orless than about 0.1° C. In some embodiments, a nucleic acidamplification reaction can be conducted under isothermal orthermal-cycling conditions.

In some embodiments, an isothermal nucleic acid amplification reactioncan be conducted for about 2, 5, 10, 15, 20, 30, 40, 50, 60 or 120minutes.

In some embodiments, an isothermal nucleic acid amplification reactioncan be conducted at about 15-25° C., or about 25-35° C., or about 35-40°C., or about 40-45° C., or about 45-50° C., or about 50-55° C., or about55-60° C.

In some embodiments, nucleic acids that have been amplified according tothe present teachings can be used in any nucleic acid sequencingworkflow, including sequencing by oligonucleotide probe ligation anddetection (e.g., SOLiD™ from Life Technologies, WO 2006/084131),probe-anchor ligation sequencing (e.g., Complete Genomics™ orPolonator™), sequencing-by-synthesis (e.g., Genetic Analyzer and HiSeq™,from Illumina), pyrophosphate sequencing (e.g., Genome Sequencer FLXfrom 454 Life Sciences), ion-sensitive sequencing (e.g., Personal GenomeMachine (PGM™) and Ion Proton™ Sequencer, both from Ion Torrent Systems,Inc.), and single molecule sequencing platforms (e.g., HeliScope™ fromHelicos™).

In some embodiments, nucleic acid that have been amplified according tothe present teachings can be sequenced by any sequencing method,including sequencing-by-synthesis, ion-based sequencing involving thedetection of sequencing byproducts using field effect transistors (e.g.,FETs and ISFETs), chemical degradation sequencing, ligation-basedsequencing, hybridization sequencing, pyrophosphate detectionsequencing, capillary electrophoresis, gel electrophoresis,next-generation, massively parallel sequencing platforms, sequencingplatforms that detect hydrogen ions or other sequencing by-products, andsingle molecule sequencing platforms. In some embodiments, a sequencingreaction can be conducted using at least one sequencing primer that canhybridize to any portion of the polynucleotide constructs, including anucleic acid adaptor or a target polynucleotide.

In some embodiments, nucleic acid amplified according to the presentteachings can be sequenced using methods that detect one or morebyproducts of nucleotide incorporation. The detection of polymeraseextension by detecting physicochemical byproducts of the extensionreaction, can include pyrophosphate, hydrogen ion, charge transfer,heat, and the like, as disclosed, for example, in U.S. Pat. No.7,948,015 to Rothberg et al.; and Rothberg et al, U.S. PatentPublication No. 2009/0026082, hereby incorporated by reference in theirentireties. Other examples of methods of detecting polymerase-basedextension can be found, for example, in Pourmand et al, Proc. Natl.Acad. Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEE ISCAS,IV-169-172; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); Sakata et al., Angew. Chem. 118:2283-2286 (2006); Esfandyapouret al., U.S. Patent Publication No. 2008/01666727; and Sakurai et al.,Anal. Chem. 64: 1996-1997 (1992).

Reactions involving the generation and detection of ions are widelyperformed. The use of direct ion detection methods to monitor theprogress of such reactions can simplify many current biological assays.For example, template-dependent nucleic acid synthesis by a polymerasecan be monitored by detecting hydrogen ions that are generated asnatural byproducts of nucleotide incorporations catalyzed by thepolymerase. Ion-sensitive sequencing (also referred to as “pH-based” or“ion-based” nucleic acid sequencing) exploits the direct detection ofionic byproducts, such as hydrogen ions, that are produced as abyproduct of nucleotide incorporation. In one exemplary system forion-based sequencing, the nucleic acid to be sequenced can be capturedin a microwell, and nucleotides can be flowed across the well, one at atime, under nucleotide incorporation conditions. The polymeraseincorporates the appropriate nucleotide into the growing strand, and thehydrogen ion that is released can change the pH in the solution, whichcan be detected by an ion sensor that is coupled with the well. Thistechnique does not require labeling of the nucleotides or expensiveoptical components, and allows for far more rapid completion ofsequencing runs. Examples of such ion-based nucleic acid sequencingmethods and platforms include the Ion Torrent PGM™ or Proton™ sequencer(Ion Torrent™ Systems, Life Technologies Corporation).

In some embodiments, target polynucleotides produced using the methods,systems and kits of the present teachings can be used as a substrate fora biological or chemical reaction that is detected and/or monitored by asensor including a field-effect transistor (FET). In various embodimentsthe FET is a chemFET or an ISFET. A “chemFET” or chemical field-effecttransistor, is a type of field effect transistor that acts as a chemicalsensor. It is the structural analog of a MOSFET transistor, where thecharge on the gate electrode is applied by a chemical process. An“ISFET” or ion-sensitive field-effect transistor, is used for measuringion concentrations in solution; when the ion concentration (such as H+)changes, the current through the transistor will change accordingly. Adetailed theory of operation of an ISFET is given in “Thirty years ofISFETOLOGY: what happened in the past 30 years and what may happen inthe next 30 years,” P. Bergveld, Sens. Actuators, 88 (2003), pp. 1-20.

In some embodiments, the FET may be a FET array. As used herein, an“array” is a planar arrangement of elements such as sensors or wells.The array may be one or two dimensional. A one dimensional array can bean array having one column (or row) of elements in the first dimensionand a plurality of columns (or rows) in the second dimension. The numberof columns (or rows) in the first and second dimensions may or may notbe the same. The FET or array can comprise 102, 103, 104, 105, 106, 107or more FETs.

In some embodiments, one or more microfluidic structures can befabricated above the FET sensor array to provide for containment and/orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) can be configured as oneor more wells (or microwells, or reaction chambers, or reaction wells,as the terms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level,and/or concentration in the given well. In some embodiments, there canbe a 1:1 correspondence of FET sensors and reaction wells.

Microwells or reaction chambers are typically hollows or wells havingwell-defined shapes and volumes which can be manufactured into asubstrate and can be fabricated using conventional microfabricationtechniques, e.g. as disclosed in the following references: Doering andNishi, Editors, Handbook of Semiconductor Manufacturing Technology,Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMSand Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al,Silicon Micromachining (Cambridge University Press, 2004); and the like.Examples of configurations (e.g. spacing, shape and volumes) ofmicrowells or reaction chambers are disclosed in Rothberg et al, U.S.patent publication 2009/0127589; Rothberg et al, U.K. patent applicationGB24611127.

In some embodiments, the biological or chemical reaction can beperformed in a solution or a reaction chamber that is in contact with,operatively coupled, or capacitively coupled to a FET such as a chemFETor an ISFET. The FET (or chemFET or ISFET) and/or reaction chamber canbe an array of FETs or reaction chambers, respectively.

In some embodiments, a biological or chemical reaction can be carriedout in a two-dimensional array of reaction chambers, wherein eachreaction chamber can be coupled to a FET, and each reaction chamber isno greater than 10 μm³ (i.e., 1 pL) in volume. In some embodiments eachreaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pLin volume. A reaction chamber can optionally be no greater than 2, 5,10, 15, 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns incross-sectional area at the top. Preferably, the array has at least 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, 10⁹, or more reaction chambers. In someembodiments, at least one of the reaction chambers is operativelycoupled to at least one of the FETs.

FET arrays as used in various embodiments according to the disclosurecan be fabricated according to conventional CMOS fabricationstechniques, as well as modified CMOS fabrication techniques and othersemiconductor fabrication techniques beyond those conventionallyemployed in CMOS fabrication. Additionally, various lithographytechniques can be employed as part of an array fabrication process.

Exemplary FET arrays suitable for use in the disclosed methods, as wellas microwells and attendant fluidics, and methods for manufacturingthem, are disclosed, for example, in U.S. Patent Publication No.20100301398; U.S. Patent Publication No. 20100300895; U.S. PatentPublication No. 20100300559; U.S. Patent Publication No. 20100197507,U.S. Patent Publication No. 20100137143; U.S. Patent Publication No.20090127589; and U.S. Patent Publication No. 20090026082, which areincorporated by reference in their entireties.

In one aspect, the disclosed methods, compositions, systems, apparatusesand kits can be used for carrying out label-free nucleic acidsequencing, and in particular, ion-based nucleic acid sequencing. Theconcept of label-free detection of nucleotide incorporation has beendescribed in the literature, including the following references that areincorporated by reference: Rothberg et al, U.S. patent publication2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470(2006). Briefly, in nucleic acid sequencing applications, nucleotideincorporations are determined by measuring natural byproducts ofpolymerase-catalyzed extension reactions, including hydrogen ions,polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase).Examples of such ion-based nucleic acid sequencing methods and platformsinclude the Ion Torrent PGM™ or Proton™ sequencer (Ion Torrent™ Systems,Life Technologies Corporation).

In some embodiments, the disclosure relates generally to methods forsequencing nucleic acids that have been amplified by the teachingsprovided herein. In one exemplary embodiment, the disclosure relatesgenerally to a method for obtaining sequence information frompolynucleotides, comprising: (a) amplifying nucleic acids; and (b)performing template-dependent nucleic acid synthesis using at least oneof the amplified nucleic acids produced during step (a) as a template.The amplifying can optionally be performed according to any of theamplification methods described herein.

In some embodiments, the template-dependent synthesis includesincorporating one or more nucleotides in a template-dependent fashioninto a newly synthesized nucleic acid strand.

Optionally, the methods can further include producing one or more ionicbyproducts of such nucleotide incorporation.

In some embodiments, the methods can further include detecting theincorporation of the one or more nucleotides into the sequencing primer.Optionally, the detecting can include detecting the release of hydrogenions.

In another embodiment, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: (a) amplifying nucleic acidsaccording to the methods disclosed herein; (b) disposing the amplifiednucleic acids into a plurality of reaction chambers, wherein one or moreof the reaction chambers are in contact with a field effect transistor(FET). Optionally, the method further includes contacting amplifiednucleic acids which are disposed into one of the reaction chambers, witha polymerase thereby synthesizing a new nucleic acid strand bysequentially incorporating one or more nucleotides into a nucleic acidmolecule. Optionally, the method further includes generating one or morehydrogen ions as a byproduct of such nucleotide incorporation.Optionally, the method further includes detecting the incorporation ofthe one or more nucleotides by detecting the generation of the one ormore hydrogen ions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET can be selected from the group consistingof: ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ orProton™ sequencer (Life Technologies), which is an ion-based sequencingsystem that sequences nucleic acid templates by detecting hydrogen ionsproduced as a byproduct of nucleotide incorporation. Typically, hydrogenions are released as byproducts of nucleotide incorporations occurringduring template-dependent nucleic acid synthesis by a polymerase. TheIon Torrent PGM™ or Proton™ sequencer detects the nucleotideincorporations by detecting the hydrogen ion byproducts of thenucleotide incorporations. The Ion Torrent PGM™ or Proton™ sequencer caninclude a plurality of nucleic acid templates to be sequenced, eachtemplate disposed within a respective sequencing reaction well in anarray. The wells of the array can each be coupled to at least one ionsensor that can detect the release of H⁺ ions or changes in solution pHproduced as a byproduct of nucleotide incorporation. The ion sensorcomprises a field effect transistor (FET) coupled to an ion-sensitivedetection layer that can sense the presence of H⁺ ions or changes insolution pH. The ion sensor can provide output signals indicative ofnucleotide incorporation which can be represented as voltage changeswhose magnitude correlates with the H⁺ ion concentration in a respectivewell or reaction chamber. Different nucleotide types can be flowedserially into the reaction chamber, and can be incorporated by thepolymerase into an extending primer (or polymerization site) in an orderdetermined by the sequence of the template. Each nucleotideincorporation can be accompanied by the release of H⁺ ions in thereaction well, along with a concomitant change in the localized pH. Therelease of H⁺ ions can be registered by the FET of the sensor, whichproduces signals indicating the occurrence of the nucleotideincorporation. Nucleotides that are not incorporated during a particularnucleotide flow may not produce signals. The amplitude of the signalsfrom the FET can also be correlated with the number of nucleotides of aparticular type incorporated into the extending nucleic acid moleculethereby permitting homopolymer regions to be resolved. Thus, during arun of the sequencer multiple nucleotide flows into the reaction chamberalong with incorporation monitoring across a multiplicity of wells orreaction chambers can permit the instrument to resolve the sequence ofmany nucleic acid templates simultaneously. Further details regardingthe compositions, design and operation of the Ion Torrent PGM™ orProton™ sequencer can be found, for example, in U.S. patent applicationSer. No. 12/002,781, now published as U.S. Patent Publication No.2009/0026082; U.S. patent application Ser. No. 12/474,897, now publishedas U.S. Patent Publication No. 2010/0137143; and U.S. patent applicationSer. No. 12/492,844, now published as U.S. Patent Publication No.2010/0282617, all of which applications are incorporated by referenceherein in their entireties.

FIG. 28 illustrates a block diagram of components of a system fornucleic acid sequencing according to an exemplary embodiment. Thecomponents include a flow cell 101 on an integrated circuit device 100,a reference electrode 108, a plurality of reagents 114 for sequencing, avalve block 116, a wash solution 110, a valve 112, a fluidics controller118, lines 120/122/126, passages 104/109/111, a waste container 106, anarray controller 124, and a user interface 128. The integrated circuitdevice 100 includes a microwell array 107 overlying a sensor array thatincludes chemical sensors as described herein. The flow cell 101includes an inlet 102, an outlet 103, and a flow chamber 105 defining aflow path of reagents over the microwell array 107.

The reference electrode 108 may be of any suitable type or shape,including a concentric cylinder with a fluid passage or a wire insertedinto a lumen of passage 111. The reagents 114 may be driven through thefluid pathways, valves, and flow cell 101 by pumps, gas pressure, orother suitable methods, and may be discarded into the waste container106 after exiting the outlet 103 of the flow cell 101. The fluidicscontroller 118 may control driving forces for the reagents 114 and theoperation of valve 112 and valve block 116 with suitable software.

The microwell array 107 includes an array of reaction regions asdescribed herein, also referred to herein as microwells, which areoperationally associated with corresponding chemical sensors in thesensor array. For example, each reaction region may be coupled to achemical sensor suitable for detecting an analyte or reaction propertyof interest within that reaction region. The microwell array 107 may beintegrated in the integrated circuit device 100, so that the microwellarray 107 and the sensor array are part of a single device or chip.

The flow cell 101 may have a variety of configurations for controllingthe path and flow rate of reagents 114 over the microwell array 107. Thearray controller 124 provides bias voltages and timing and controlsignals to the integrated circuit device 100 for reading the chemicalsensors of the sensor array. The array controller 124 also provides areference bias voltage to the reference electrode 108 to bias thereagents 114 flowing over the microwell array 107.

During an experiment, the array controller 124 collects and processesoutput signals from the chemical sensors of the sensor array throughoutput ports on the integrated circuit device 100 via bus 127. The arraycontroller 124 may be a computer or other computing means. The arraycontroller 124 may include memory for storage of data and softwareapplications, a processor for accessing data and executing applications,and components that facilitate communication with the various componentsof the system in FIG. 28 .

The values of the output signals of the chemical sensors indicatephysical and/or chemical parameters of one or more reactions takingplace in the corresponding reaction regions in the microwell array 107.For example, in an exemplary embodiment, the values of the outputsignals may be processed using the techniques disclosed in Rearick etal., U.S. patent application Ser. No. 13/339,846, filed Dec. 29, 2011,based on U.S. Prov. Pat. Appl. Nos. 61/428,743, filed Dec. 30, 2010, and61/429,328, filed Jan. 3, 2011, and in Hubbell, U.S. patent applicationSer. No. 13/339,753, filed Dec. 29, 2011, based on U.S. Prov. Pat. Appl.No. 61/428,097, filed Dec. 29, 2010, which are all incorporated byreference herein in their entirety.

The user interface 128 may display information about the flow cell 101and the output signals received from chemical sensors in the sensorarray on the integrated circuit device 100. The user interface 128 mayalso display instrument settings and controls, and allow a user to enteror set instrument settings and controls.

In an exemplary embodiment, during the experiment the fluidicscontroller 118 may control delivery of the individual reagents 114 tothe flow cell 101 and integrated circuit device 100 in a predeterminedsequence, for predetermined durations, at predetermined flow rates. Thearray controller 124 can then collect and analyze the output signals ofthe chemical sensors indicating chemical reactions occurring in responseto the delivery of the reagents 114.

During the experiment, the system may also monitor and control thetemperature of the integrated circuit device 100, so that reactions takeplace and measurements are made at a known predetermined temperature.

The system may be configured to let a single fluid or reagent contactthe reference electrode 108 throughout an entire multi-step reactionduring operation. The valve 112 may be shut to prevent any wash solution110 from flowing into passage 109 as the reagents 114 are flowing.Although the flow of wash solution may be stopped, there may still beuninterrupted fluid and electrical communication between the referenceelectrode 108, passage 109, and the microwell array 107. The distancebetween the reference electrode 108 and the junction between passages109 and 111 may be selected so that little or no amount of the reagentsflowing in passage 109 and possibly diffusing into passage 111 reach thereference electrode 108. In an exemplary embodiment, the wash solution110 may be selected as being in continuous contact with the referenceelectrode 108, which may be especially useful for multi-step reactionsusing frequent wash steps.

FIG. 29 illustrates cross-sectional and expanded views of a portion ofthe integrated circuit device 100 and flow cell 101. During operation,the flow chamber 105 of the flow cell 101 confines a reagent flow 208 ofdelivered reagents across open ends of the reaction regions in themicrowell array 107. The volume, shape, aspect ratio (such as basewidth-to-well depth ratio), and other dimensional characteristics of thereaction regions may be selected based on the nature of the reactiontaking place, as well as the reagents, byproducts, or labelingtechniques (if any) that are employed.

The chemical sensors of the sensor array 205 are responsive to (andgenerate output signals) chemical reactions within associated reactionregions in the microwell array 107 to detect an analyte or reactionproperty of interest. The chemical sensors of the sensor array 205 mayfor example be chemically sensitive field-effect transistors (chemFETs),such as ion-sensitive field effect transistors (ISFETs). Examples ofchemical sensors and array configurations that may be used inembodiments are described in U.S. Patent Application Publication No.2010/0300559, No. 2010/0197507, No. 2010/0301398, No. 2010/0300895, No.2010/0137143, and No. 2009/0026082, and U.S. Pat. No. 7,575,865, eachwhich are incorporated by reference herein.

FIG. 30 illustrates a cross-sectional view of two representativechemical sensors and their corresponding reaction regions according toan exemplary embodiment. In FIG. 30 , two chemical sensors 350, 351 areshown, representing a small portion of a sensor array that can includemillions of chemical sensors. In some embodiments, the sensor array caninclude at least 1 million chemical sensors and optionally at least 1million corresponding reaction regions, at least 10 million chemicalsensors and optionally at least 10 million corresponding reactionregions, at least 100 million chemical sensors and optionally at least100 million corresponding reaction regions, at least 500 millionchemical sensors and optionally at least 500 million correspondingreaction regions, or even at least 1 billion chemical sensors andoptionally at least 1 billion corresponding reaction regions.

Chemical sensor 350 is coupled to corresponding reaction region 301, andchemical sensor 351 is coupled to corresponding reaction region 302.Chemical sensor 350 is representative of the chemical sensors in thesensor array. In the illustrated example, the chemical sensor 350 is anion-sensitive field effect transistor. The chemical sensor 350 includesa floating gate structure 318 having a floating gate conductor (referredto herein as the sensor plate 320) separated from the reaction region301 by sensing material 316. As shown in FIG. 30 , the sensor plate 320is the uppermost patterned layer of conductive material in the floatinggate structure 318 underlying the reaction region 301.

In the illustrated example, the floating gate structure 318 includesmultiple patterned layers of conductive material within layers ofdielectric material 319. As described in more detail below, the uppersurface of the sensing material 316 acts as the sensing surface 317 forthe chemical sensor 350.

In the illustrated embodiment, the sensing material 316 is anion-sensitive material, such that the presence of ions or other chargedspecies in a solution in the reaction region 301 alters the surfacepotential of the sensing surface 317. The change in the surfacepotential is due to the protonation or deprotonation of surface chargegroups at the sensing surface caused by the ions present in thesolution. The sensing material 316 may be deposited using varioustechniques, or naturally formed during one or more of the manufacturingprocesses used to form the chemical sensor 350. In some embodiments, thesensing material 316 is a metal oxide, such as an oxide of silicon,tantalum, aluminum, lanthanum, titanium, zirconium, hafnium, tungsten,palladium, iridium, etc.

In some embodiments, the sensing material 316 is an oxide of the upperlayer of conductive material of the sensor plate 320. For example, theupper layer of the sensor plate 320 may be titanium nitride, and thesensing material 316 may comprise titanium oxide or titanium oxynitride.More generally, the sensing material 316 may comprise one or more of avariety of different materials to facilitate sensitivity to particularions. For example, silicon nitride or silicon oxynitride, as well asmetal oxides such as silicon oxide, aluminum or tantalum oxides,generally provide sensitivity to hydrogen ions, whereas sensingmaterials comprising polyvinyl chloride containing valinomycin providesensitivity to potassium ions. Materials sensitive to other ions such assodium, silver, iron, bromine, iodine, calcium, and nitrate may also beused, depending upon the implementation.

The chemical sensor 350 also includes a source region 321 and a drainregion 322 within a semiconductor substrate 354. The source region 321and the drain region 322 comprise doped semiconductor material have aconductivity type different from the conductivity type of the substrate354. For example, the source region 321 and the drain region 322 maycomprise doped P-type semiconductor material, and the substrate maycomprise doped N-type semiconductor material.

Channel region 323 separates the source region 321 and the drain region322. The floating gate structure 318 overlies the channel region 323,and is separated from the substrate 354 by a gate dielectric 352. Thegate dielectric 352 may be for example silicon dioxide. Alternatively,other dielectrics may be used for the gate dielectric 352.

As shown in FIG. 30 , the reaction region 301 extends through a fillmaterial 310 on the dielectric material 319. The fill material 310 mayfor example comprise one or more layers of dielectric material, such assilicon dioxide or silicon nitride.

The dimensions (e.g. the width and depth) of the reaction regions 301,302, and their pitch (the center-to-center distance between adjacentreaction regions), can vary from implementation to implementation. Insome embodiments, the reaction regions can have a characteristicdiameter, defined as the square root of 4 times the plan viewcross-sectional area (A) divided by Pi (e.g., sqrt(4*A/π), of notgreater than 5 micrometers, such as not greater than 3.5 micrometers,not greater than 2.0 micrometers, not greater than 1.6 micrometers, notgreater than 1.0 micrometers, not greater than 0.8 micrometers, notgreater than 0.6 micrometers, not greater than 0.4 micrometers, notgreater than 0.2 micrometers or even not greater than 0.1 micrometers.

In some embodiments, the pitch between adjacent reaction regions is notgreater than 10 micrometers, not greater than 5 micrometers, not greaterthan 2 micrometers, not greater than 1 micrometer, or even not greaterthan 0.5 micrometers.

In the illustrated embodiment, the reaction regions 301, 302 areseparated by a distance that that is equal to their width.Alternatively, the separation distance between adjacent reaction regionsmay be less than their width. For example, the separation distance maybe a minimum feature size for the process (e.g. a lithographic process)used to form the reaction regions 301, 302. In such a case, theseparation distance may be significantly less than the width ofindividual reaction regions.

The sensor plate 320, the sensing material 316 and the reaction region301 may for example have circular cross-sections. Alternatively, thesemay be non-circular. For example, the cross-section may be square,rectangular, hexagonal, or irregularly shaped.

The device in FIG. 30 can also include additional elements such as arraylines (e.g. word lines, bit lines, etc.) for accessing the chemicalsensors, additional doped regions in the substrate 354, and othercircuitry (e.g. access circuitry, bias circuitry etc.) used to operatethe chemical sensors, depending upon the device and array configurationin which the chemical sensors described herein are implemented. In someembodiments, the device may for example be manufactured using techniquesdescribed in U.S. Patent Application Publication No. 2010/0300559, No.2010/0197507, No. 2010/0301398, No. 2010/0300895, No. 2010/0137143, andNo. 2009/0026082, and U.S. Pat. No. 7,575,865, each which areincorporated by reference herein.

In operation, reactants, wash solutions, and other reagents may move inand out of the reaction region 301 by a diffusion mechanism 340. Thechemical sensor 350 is responsive to (and generates an output signalrelated to) the amount of a charge 324 present on the sensing material316 opposite the sensor plate 320. Changes in the charge 324 causechanges in the voltage on the floating gate structure 318, which in turnchanges in the threshold voltage of the transistor. This change inthreshold voltage can be measured by measuring the current in thechannel region 323 between the source region 321 and a drain region 322.As a result, the chemical sensor 350 can be used directly to provide acurrent-based output signal on an array line connected to the sourceregion 321 or drain region 322, or indirectly with additional circuitryto provide a voltage-based output signal.

In an embodiment, reactions carried out in the reaction region 301 canbe analytical reactions to identify or determine characteristics orproperties of an analyte of interest. Such reactions can generatedirectly or indirectly byproducts that affect the amount of chargeadjacent to the sensor plate 320. If such byproducts are produced insmall amounts or rapidly decay or react with other constituents,multiple copies of the same analyte may be analyzed in the reactionregion 301 at the same time in order to increase the output signalgenerated. In an embodiment, multiple copies of an analyte may beattached to a solid phase support 312, either before or after depositioninto the reaction region 301. The solid phase support 312 may bemicroparticles, nanoparticles, beads, solid or porous comprising gels,or the like. For simplicity and ease of explanation, solid phase support312 is also referred herein as a particle. For a nucleic acid analyte,multiple, connected copies may be made by rolling circle amplification(RCA), exponential RCA, Recombinase Polymerase Amplification (RPA),Polymerase Chain Reaction amplification (PCR), emulsion PCRamplification, or like techniques, to produce an amplicon without theneed of a solid support.

In various exemplary embodiments, the methods, systems, and computerreadable media described herein may advantageously be used to processand/or analyze data and signals obtained from electronic orcharged-based nucleic acid sequencing. In electronic or charged-basedsequencing (such as, pH-based sequencing), a nucleotide incorporationevent may be determined by detecting ions (e.g., hydrogen ions) that aregenerated as natural by-products of polymerase-catalyzed nucleotideextension reactions. This may be used to sequence a sample or templatenucleic acid, which may be a fragment of a nucleic acid sequence ofinterest, for example, and which may be directly or indirectly attachedas a clonal population to a solid support, such as a particle,microparticle, bead, etc. The sample or template nucleic acid may beoperably associated to a primer and polymerase and may be subjected torepeated cycles or “flows” of nucleotide addition (which may be referredto herein as “nucleotide flows” from which nucleotide incorporations mayresult) and washing. The primer may be annealed to the sample ortemplate so that the primer's 3′ end can be extended by a polymerasewhenever nucleotides complementary to the next base in the template areadded. Then, based on the known sequence of nucleotide flows and onmeasured output signals of the chemical sensors indicative of ionconcentration during each nucleotide flow, the identity of the type,sequence and number of nucleotide(s) associated with a sample nucleicacid present in a reaction region coupled to a chemical sensor can bedetermined.

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations can be detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, templates, optionally pre-boundto a sequencing primer and/or a polymerase, can be loaded into reactionchambers (such as the microwells disclosed in Rothberg et al, citedherein), after which repeated cycles of nucleotide addition and washingcan be carried out. In some embodiments, such templates can be attachedas clonal populations to a solid support, such as particles, bead, orthe like, and said clonal populations are loaded into reaction chambers.

In another embodiment, the templates, optionally bound to a polymerase,are distributed, deposited or positioned to different sites of thearray. The site of the array include primers and the methods can includehybridizing different templates to the primers within different sites.

In each addition step of the cycle, the polymerase can extend the primerby incorporating added nucleotide only if the next base in the templateis the complement of the added nucleotide. If there is one complementarybase, there is one incorporation, if two, there are two incorporations,if three, there are three incorporations, and so on. With each suchincorporation there is a hydrogen ion released, and collectively apopulation of templates releasing hydrogen ions changes the local pH ofthe reaction chamber. The production of hydrogen ions is monotonicallyrelated to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, can be proportional tothe number of contiguous identical complementary bases. If the next basein the template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepcan be performed, in which an unbuffered wash solution at apredetermined pH is used to remove the nucleotide of the previous stepin order to prevent misincorporations in later cycles. In someembodiments, the after each step of adding a nucleotide, an additionalstep can be performed wherein the reaction chambers are treated with anucleotide-destroying agent, such as apyrase, to eliminate any residualnucleotides remaining in the chamber, which may result in spuriousextensions in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction can beexposed to the different nucleotides one at a time. For example,nucleotides can be added in the following sequence: dATP, dCTP, dGTP,dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed bya wash step. The cycles may be repeated for 50 times, 100 times, 200times, 300 times, 400 times, 500 times, 750 times, or more, depending onthe length of sequence information desired.

In some embodiments, sequencing can be performed according to the userprotocols supplied with the PGM™ or Proton™ sequencer. Example 3provides one exemplary protocol for ion-based sequencing using the IonTorrent PGM™ sequencer (Ion Torrent™ Systems, Life Technologies, CA).

In some embodiments, the disclosure relates generally to methods forsequencing a population of template polynucleotides, comprising: (a)generating a plurality of amplicons by clonally amplifying a pluralityof template polynucleotides onto a plurality of surfaces, wherein theamplifying is performed within a single continuous phase of a reactionmixture and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% of the resulting amplicons are substantially monoclonal innature. In some embodiments, a sufficient number of substantiallymonoclonal amplicons are produced in a single amplification reaction togenerate at least 100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1 GBor 2 GB of AQ20 sequencing reads on an Ion Torrent PGM™ 314, 316 or 318sequencer. The term “AQ20 and its variants, as used herein, refers to aparticular method of measuring sequencing accuracy in the Ion TorrentPGM™ sequencer. Accuracy can be measured in terms of the Phred-like Qscore, which measures accuracy on logarithmic scale that: Q10=90%,Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. For example, in aparticular sequencing reaction, accuracy metrics can be calculatedeither through prediction algorithms or through actual alignment to aknown reference genome. Predicted quality scores (“Q scores”) can bederived from algorithms that look at the inherent properties of theinput signal and make fairly accurate estimates regarding if a givensingle base included in the sequencing “read” will align. In someembodiments, such predicted quality scores can be useful to filter andremove lower quality reads prior to downstream alignment. In someembodiments, the accuracy can be reported in terms of a Phred-like Qscore that measures accuracy on logarithmic scale such that: Q10=90%,Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. In someembodiments, the data obtained from a given polymerase reaction can befiltered to measure only polymerase reads measuring “N” nucleotides orlonger and having a Q score that passes a certain threshold, e.g., Q10,Q17, Q100 (referred to herein as the “NQ17” score). For example, the100Q20 score can indicate the number of reads obtained from a givenreaction that are at least 100 nucleotides in length and have Q scoresof Q20 (99%) or greater. Similarly, the 200Q20 score can indicate thenumber of reads that are at least 200 nucleotides in length and have Qscores of Q20 (99%) or greater.

In some embodiments, the accuracy can also be calculated based on properalignment using a reference genomic sequence, referred to herein as the“raw” accuracy. This is single pass accuracy, involving measurement ofthe “true” per base error associated with a single read, as opposed toconsensus accuracy, which measures the error rate from the consensussequence which is the result of multiple reads. Raw accuracymeasurements can be reported in terms of “AQ” scores (for alignedquality). In some embodiments, the data obtained from a given polymerasereaction can be filtered to measure only polymerase reads measuring “N”nucleotides or longer having a AQ score that passes a certain threshold,e.g., AQ10, AQ17, AQ100 (referred to herein as the “NAQ17” score). Forexample, the 100AQ20 score can indicate the number of reads obtainedfrom a given polymerase reaction that are at least 100 nucleotides inlength and have AQ scores of AQ20 (99%) or greater. Similarly, the200AQ20 score can indicate the number of reads that are at least 200nucleotides in length and have AQ scores of AQ20 (99%) or greater.

In some embodiments, the present teachings provide systems for nucleicacid amplification, comprising any combination of: beads attached with aplurality of a first primer, second primer, third primer,polynucleotides, recombinase, recombinase loading protein,single-stranded binding protein (SSB), polymerase, nucleotides, ATP,phosphocreatine, creatine kinase, hybridization solutions, and/orwashing solutions. A system can include all or some of these components.In some embodiments, systems for nucleic acid amplification can furthercomprise any combination of: buffers and/or cations (e.g., divalentcations).

In some embodiments, the present teachings provide kits for nucleic acidamplification. In some embodiments, kits include any reagent that can beused for nucleic acid amplification. In some embodiments, kits includeany combination of: beads attached with a plurality of a first primer,second primer, third primer, polynucleotides, recombinase, recombinaseloading protein, single-stranded binding protein (SSB), polymerase,nucleotides, ATP, phosphocreatine, creatine kinase, hybridizationsolutions, washing solutions, buffers and/or cations (e.g., divalentcations). A kit can include all or some of these components.

In some embodiments, the disclosure relates generally to methods,compositions, systems useful for amplifying different nucleic acidtemplates in parallel in a plurality of compartmentalized reactionvolumes, as opposed to amplification within a single continuous liquidphase. For example, the nucleic acid templates can be distributed ordeposited into an array of reaction chambers, or an array of reactionvolumes, such that at least two such chambers or volumes in the arrayeach receive a single nucleic acid template. In some embodiments, aplurality of separate reaction volumes is formed. The reaction chambers(or reaction volumes) can optionally be sealed prior to amplification.In another embodiment, the reaction mixture can be compartmentalized orseparated into a plurality of microreactors dispersed within acontinuous phase of an emulsion. The compartmentalized or separatereaction volumes optionally do not mix or communicate, or are notcapable of mixing or communicating, with each other. In someembodiments, at least some of the reaction chambers (or reactionvolumes) include a recombinase, and optionally a polymerase. Thepolymerase can be a strand-displacing polymerase.

In some embodiments, the disclosure relates generally to compositions,systems, methods, apparatuses and kits for nucleic acid synthesis and/oramplification including emulsions. As used herein, the term “emulsion”includes any composition including a mixture of a first liquid and asecond liquid, wherein the first and second liquids are substantiallyimmiscible with each other. Typically, one of the liquids is hydrophilicwhile the other liquid is hydrophobic. Typically, the emulsion includesa dispersed phase and a continuous phase. For example, the first liquidcan form a dispersed phase that is dispersed in the second liquid, whichforms the continuous phase. The dispersed phase is optionally comprisedpredominantly of the first liquid. The continuous phase is optionallycomprised predominantly of the second liquid. In various embodiments,the same two liquids can form different types of emulsions. For example,in a mixture including both oil and water can form, firstly, anoil-in-water emulsion, where the oil is the dispersed phase, and wateris the dispersion medium. Secondly, they can form a water-in-oilemulsion, where water is the dispersed phase and oil is the externalphase. Multiple emulsions are also possible, including a“water-in-oil-in-water” emulsion and an “oil-in-water-in-oil” emulsion.In some embodiments, the dispersed phase includes one or moremicroreactors in which nucleic acid templates can be individuallyamplified. One or more microreactors can form compartmentalized reactionvolumes in which separate amplification reactions can occur. One exampleof a suitable vehicle for nucleic acid amplification includes awater-in-oil emulsion wherein the water-based phase includes severalaqueous microreactors that are dispersed within an oil phase of anemulsion. In some embodiments, the emulsion can further include anemulsifier or surfactant. The emulsifier or surfactant can be useful instabilizing the emulsion under nucleic acid synthesis conditions.

In some embodiments, the disclosure relates generally to a compositioncomprises an emulsion including a reaction mixture. The emulsion caninclude an aqueous phase. The aqueous phase can be dispersed in acontinuous phase of the emulsion. The aqueous phase can include one ormore microreactors. In some embodiments, the reaction mixture iscontained in a plurality of liquid phase microreactors within a phase ofan emulsion. Optionally, the reaction mixture includes a recombinase.Optionally, the reaction mixture includes a plurality of differentpolynucleotides. Optionally, the reaction mixture includes a pluralityof supports. Optionally, the reaction mixture includes any combinationof a recombinase, a plurality of different polynucleotides and/or aplurality of supports. Optionally, at least one of the supports can beattached to a substantially monoclonal nucleic acid population.

In some embodiments, the disclosure relates generally to a compositioncomprising a reaction mixture, the reaction mixture including (i) aplurality of supports, (ii) a plurality of different polynucleotides and(iii) a recombinase, the reaction mixture contained in a plurality ofliquid phase microreactors in an emulsion.

In some embodiments, the disclosure relates generally to a compositioncomprising a reaction mixture, the reaction mixture including (i) arecombinase and (ii) a plurality of supports, at least one of thesupports being attached to a substantially monoclonal nucleic acidpopulation, wherein the reaction mixture is contained in a plurality ofliquid phase microreactors in an emulsion.

Optionally, the emulsion includes a hydrophilic phase.

Optionally, the emulsion comprises a hydrophilic phase dispersed in ahydrophobic phase. For example, the emulsion can include a water-in-oilemulsion.

In some embodiments, the hydrophilic phase includes a plurality ofmicroreactors.

Optionally, the reaction mixture is contained in a single reactionvessel.

Optionally, the sequences of the plurality of different polynucleotidescan be the same or different.

Optionally, at least one of the plurality of supports is linked to aplurality of first primers (e.g., forward amplification primers).

Optionally, the reaction mixture further includes a plurality of asecond primer (e.g., reverse amplification primers).

In some embodiments, at least one of the plurality of supports furtherincludes a plurality of second primers.

In some embodiments, at least one of the plurality of supports includesa plurality of first and second primers.

In some embodiments, the first and second primers comprise the samesequences. In some embodiments, the first and second primers comprisedifferent sequences.

In some embodiments, the support comprises a bead, particle, a planarsurface, or an interior wall of a channel or tube.

In some embodiments, the reaction mixture further includes a polymeraseand a plurality of nucleotides.

In some embodiments, the disclosure relates generally to a compositioncomprising an emulsion. Optionally, the emulsion comprises a hydrophilicphase and a hydrophobic phase. Optionally, the emulsion comprises ahydrophilic phase dispersed in a hydrophobic phase. Optionally, thehydrophilic phase can include any combination of a plurality ofpolynucleotide templates, a plurality of supports and/or a recombinase.Optionally, the hydrophilic phase can include a plurality ofpolynucleotide templates. Optionally, the hydrophilic phase can includea plurality of supports. Optionally, the hydrophilic phase can include arecombinase.

In some embodiments, a composition comprises an emulsion comprising ahydrophilic phase and a hydrophobic phase, wherein the hydrophilic phaseincludes a plurality of polynucleotide templates, a plurality ofsupports and a recombinase.

In some embodiments, the disclosure relates generally to a compositioncomprises an emulsion including a hydrophilic phase dispersed in ahydrophobic phase. Optionally, the hydrophilic phase includes aplurality of microreactors. Optionally, at least two microreactors ofthe plurality includes a different polynucleotide template. Optionally,the sequences of the different polynucleotide templates is the same ordifferent. Optionally, a first microreactor includes a firstpolynucleotide template and a second microreactor includes a secondpolynucleotide template. Optionally, the first and the secondpolynucleotide templates comprise the same or different sequences.Optionally, at least two microreactors of the plurality includes arecombinase.

In some embodiments, a composition comprises an emulsion including ahydrophilic phase dispersed in a hydrophobic phase, wherein thehydrophilic phase including a plurality of microreactors, at least twomicroreactors of the plurality including a different polynucleotidetemplate and a recombinase.

In some embodiments, the hydrophilic phase includes a plurality ofaqueous microreactors, at least two of the microreactors each includinga different polynucleotide template, a support, and a recombinase.

Optionally, a first microreactor includes a first polynucleotidetemplate and a second microreactor includes a second polynucleotidetemplate. Optionally, the first and the second polynucleotide templatescomprise the same or different sequences.

Optionally, at least one of the plurality of supports is linked to aplurality of first primers (e.g., forward amplification primers).

Optionally, the reaction mixture further includes a plurality of asecond primer (e.g., reverse amplification primers).

In some embodiments, at least one of the plurality of supports furtherincludes a plurality of second primers.

In some embodiments, at least one of the plurality of supports includesa plurality of first and second primers.

In some embodiments, the first and second primers comprise the samesequences.

In some embodiments, the first and second primers comprise differentsequences. In some embodiments, the hydrophilic phase further includes apolymerase.

In some embodiments, the polymerase comprises a strand displacingpolymerase. In some embodiments, the hydrophilic phase includesnucleotides.

In some embodiments, the disclosure relates generally to methods (aswell as associated compositions and systems) for nucleic acid synthesis,comprising: (a) forming a reaction mixture; and (b) subjecting thereaction mixture to amplification conditions. Optionally, the reactionmixture is contained within a hydrophilic phase of an emulsion.Optionally, the emulsion includes a hydrophilic phase and a hydrophobicphase. Optionally, the emulsion comprises a hydrophilic phase dispersedin a hydrophobic phase. Optionally, the reaction mixture contains anycombination of a plurality of supports, a plurality of differentpolynucleotides and/or a recombinase. Optionally, the reaction mixturecontains a plurality of supports. Optionally, the reaction mixturecontains a plurality of different polynucleotides. Optionally, thesequences of the different polynucleotide templates is the same ordifferent. Optionally, a first microreactor includes a firstpolynucleotide template and a second microreactor includes a secondpolynucleotide template. Optionally, the first and the secondpolynucleotide templates comprise the same or different sequences.Optionally, the reaction mixture contains a recombinase. Optionally, theamplification conditions include isothermal or thermo-cyclingtemperature conditions. Optionally, the method further includes formingat least two supports subjecting the emulsion to amplificationconditions results in forming a plurality of supports, wherein at leasttwo of the supports are each independently attached to a substantiallymonoclonal nucleic acid population.

In some embodiments, the disclosure relates generally to methods (aswell as associated compositions and systems) for nucleic acid synthesis,comprising: (a) forming a reaction mixture containing a plurality ofsupports, a plurality of different polynucleotides and a recombinase,the reaction mixture contained within a hydrophilic phase of anemulsion; and (b) subjecting the emulsion including the reaction mixtureto isothermal amplification conditions, thereby generating a pluralityof supports and a substantially monoclonal nucleic acid populationattached thereto.

In some embodiments, the emulsion includes a water-in-oil emulsion. Insome embodiments, the liquid phase microreactors comprise a hydrophilicphase. In some embodiments, the emulsion comprises a hydrophilic phasedispersed in a hydrophobic phase. In some embodiments, the reactionmixture is formed in a single reaction vessel. Optionally, the sequencesof the plurality of different polynucleotide templates is the same ordifferent. Optionally, a first polynucleotide template includes a firstsequence and a second polynucleotide template includes a second sequenceOptionally, the first and the second polynucleotide template sequencesare the same or different. Optionally, at least one of the plurality ofsupports is linked to a plurality of first primers (e.g., forwardamplification primers). Optionally, the reaction mixture furtherincludes a plurality of a second primer (e.g., reverse amplificationprimers). In some embodiments, at least one of the plurality of supportsfurther includes a plurality of second primers. In some embodiments, atleast one of the plurality of supports includes a plurality of first andsecond primers. In some embodiments, the first and second primerscomprise the same sequences. In some embodiments, the first and secondprimers comprise different sequences. In some embodiments, the nucleicacid synthesis method further includes recovering from the reactionmixture at least some of the supports attached to substantially nucleicacid monoclonal populations. In some embodiments, the nucleic acidsynthesis method further includes depositing onto a surface at leastsome of the supports attached to the substantially monoclonal nucleicacid populations. In some embodiments, the nucleic acid synthesis methodfurther includes forming an array by depositing onto a surface at leastsome of the supports attached to the substantially monoclonal nucleicacid populations. In some embodiments, the nucleic acid synthesis methodfurther includes sequencing at least one substantially monoclonalnucleic acid population attached to the support. In some embodiments,the support comprises a bead, particle, a planar surface, or an interiorwall of a channel or tube. In some embodiments, the reaction mixturefurther includes a polymerase and a plurality of nucleotides. In someembodiments, the polymerase comprises a strand displacing polymerase.

In some embodiments, methods for nucleic acid synthesis comprise formingan emulsion. Optionally, the emulsion comprises a hydrophilic phase anda hydrophobic phase. Optionally, the emulsion comprises a hydrophilicphase dispersed in a hydrophobic phase. Optionally, the hydrophilicphase includes a plurality of microreactors. Optionally, at least twomicroreactors of the plurality include individual polynucleotidetemplates. Optionally, at least two microreactors of the pluralityinclude a different polynucleotide template. Optionally, a firstmicroreactor includes a first polynucleotide template, and a secondmicroreactor includes a second polynucleotide template. Optionally, thefirst and the second polynucleotide templates have the same or differentsequences. Optionally, at least two microreactors of the pluralityincluding a recombinase.

In some embodiments, the disclosure relates generally to methods (aswell as associated compositions and systems) for nucleic acid synthesis,comprising: forming an emulsion including a hydrophilic phase dispersedin a hydrophobic phase, the hydrophilic phase including a plurality ofmicroreactors, at least two microreactors of the plurality including adifferent polynucleotide template and a recombinase.

In some embodiments, the emulsion includes a water-in-oil emulsion. Insome embodiments, the hydrophilic phase further includes a polymerase.In some embodiments, the polymerase is a strand displacing polymerase.In some embodiments, the hydrophilic phase includes nucleotides. In someembodiments, the emulsion is formed in a single reaction vessel.Optionally, the sequences of the different polynucleotide templates arethe same or different. Optionally, a first microreactor includes a firstpolynucleotide template and a second microreactor includes a secondpolynucleotide template. Optionally, the first and the secondpolynucleotide templates comprise the same or different sequences. Insome embodiments, the at least two microreactors of the pluralityinclude a plurality of supports. Optionally, at least one of theplurality of supports is linked to a plurality of first primers (e.g.,forward amplification primers). Optionally, the reaction mixture furtherincludes a plurality of a second primer (e.g., reverse amplificationprimers). In some embodiments, at least one of the plurality of supportsfurther includes a plurality of second primers. In some embodiments, atleast one of the plurality of supports includes a plurality of first andsecond primers. In some embodiments, the first and second primerscomprise the same sequences. In some embodiments, the first and secondprimers comprise different sequences. In some embodiments, thehydrophilic phase includes a reaction mixture. In some embodiments, thereaction mixture comprises a plurality of polynucleotide templates, aplurality of supports and a recombinase. In some embodiments, methodsfor nucleic acid synthesis further comprise subjecting the emulsion(e.g., including the reaction mixture) to isothermal amplificationconditions, thereby generating a plurality of substantially monoclonalnucleic acid populations. In some embodiments, the plurality ofsubstantially monoclonal nucleic acid populations are attached to theplurality of supports. In some embodiments, the nucleic acid synthesismethod further includes recovering from the reaction mixture at leastsome of the supports attached to substantially nucleic acid monoclonalpopulations. In some embodiments, the nucleic acid synthesis methodfurther includes depositing onto a surface at least some of the supportsattached to the substantially monoclonal nucleic acid populations. Insome embodiments, the nucleic acid synthesis method further includesforming an array by depositing onto a surface at least some of thesupports attached to the substantially monoclonal nucleic acidpopulations. In some embodiments, the nucleic acid synthesis methodfurther includes sequencing at least one substantially monoclonalnucleic acid population attached to the support. In some embodiments,the support comprises a bead, particle, a planar surface, or an interiorwall of a channel or tube. In some embodiments, the reaction mixturefurther includes a polymerase and a plurality of nucleotides. In someembodiments, the polymerase comprises a strand displacing polymerase.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems and kits) for nucleic acidsynthesis, comprising providing a double stranded nucleic acid template;and performing template-dependent nucleotide polymerization using apolymerase, optionally under isothermal or substantially isothermalconditions. In some embodiments, the method further includes forming asubstantially monoclonal nucleic acid population. In some embodiments,the methods include contacting a double-stranded nucleic acid templatewith a single reaction mixture including reagents for nucleic acidsynthesis. In some embodiments, the reaction mixture includes all of thecomponents required to perform recombinase polymerase amplification(RPA) or template walking. Optionally, the RPA reaction mixture canfurther include one or more additional polymerases for nucleic acidamplification. In some embodiments, the one or more additionalpolymerases include a polymerase with reduced 3′-5′ exonucleaseactivity.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems, and kits) for nucleic acidamplification. In some embodiments, the methods for nucleic acidamplification include subjecting the double-stranded nucleic acidtemplate to conditions such that the double-stranded nucleic acidtemplate forms substantially single-stranded nucleic acid templatessuitable for nucleic acid amplification. Such conditions can include,for example, thermal denaturation or chemical denaturation, or both. Insome embodiments, methods for nucleic acid synthesis and/or nucleic acidamplification can include amplifying a double stranded (or at leastpartially double stranded) template without subjecting the template tothermal denaturation or chemical denaturation.

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems, and kits) for nucleic acidamplification and downstream sequencing. In some embodiments, the methodfor nucleic acid amplification is compatible with DNA and/or RNAsequencing. In one embodiment, template nucleic acids, such as mRNA, canbe transcribed to cDNA using reverse-transcriptase (see for exampleRT-PCR kits commercially available from New England Biolabs (MA) or LifeTechnologies Corporation (CA)), and applied as template DNA in thenucleic acid amplification method. In one embodiment, methods fornucleic acid amplification and downstream sequencing can includesequencing via non-optical sequencing means such as sequencing performedusing a Personal Genome Machine (PGM) or Proton Sequencer (LifeTechnologies Corporation, CA). In some embodiments, methods for nucleicacid amplification and sequencing can include sequencing via opticalsequencing means such as SOLiD sequencing (Life TechnologiesCorporation, CA) or MiSeq/HiSeq sequencing (Illumina, Calif.). In someembodiments, methods for nucleic acid amplification and downstreamsequencing can be performed using any next generation sequencingplatform.

In some embodiments, methods for nucleic acid amplification can includebridge or cluster polymerase chain reaction (PCR). Bridge PCR refers toan amplification technique in which all of the primers required foramplification (e.g., forward and reverse primers) are attached to one ormore surfaces, thereby limiting amplification to two-dimensionalsurfaces (See Adessi et al., (2000) Nucleic Acids Res. 15:28; Mercier etal., Biophysical Journal (2003) 85:2075-2086). In some embodiments, the5′ end of the primers is covalently attached to the surface (e.g.,silica, slide) instead of freely diffusing in the amplification reactionmixture. In some embodiments, bridge amplification can occur as atwo-step process. First, a freely diffusing DNA template is captured byan immobilized primer attached to the surface and the template DNA iscopied by a polymerase. The initial DNA template is released back intothe amplification reaction mixture after annealing and extension of theattached primer; while the copy of the DNA template remains attached tothe surface. In some embodiments, a wash step is included after thisinitial process. Second, once a copy of the DNA template is attached tothe surface, the free end of the attached DNA template copy canhybridize to a primer attached to the surface that is complementary tothe DNA template copy and subsequent amplification of the DNA templatecopy can occur. In this second process, a colony of localized DNAtemplate molecules that are identical to the initial DNA template can beformed.

In some embodiments, methods of nucleic acid amplification can includepolony PCR. While being similar to the overall concept of amplificationby bridge PCR, polony PCR uses a different means of primer attachmentand different amplification conditions (See Shendure et al., (2005)Science 309:1728). In some embodiments, polony PCR includes a pluralityof beads having pre-attached primers (e.g., forward primers) that arecomplementary to the DNA template to act as the surface foramplification. In some embodiments, the beads also include reverseprimers to provide a means to amplify both the forward and reversestrands of the amplified template DNA. In some embodiments, theamplification reaction conditions include emulsion based PCR conditions.In some embodiments, components of the amplification reaction mixturecan contain additional immobilization characteristics. For example, thepolynucleotide template and/or amplification primers can be suspended ingels or other matrices during amplification to prevent migration of theamplification reaction products from the site of synthesis. Such gelsand matrices typically require removal prior to downstream processing,requiring the use of appropriate “melting” or other recovery steps.

In another embodiment, the disclosure provides methods for performingsubstantially clonal amplification of multiple polynucleotide templatesin parallel in a single continuous liquid phase of a reaction mixture,without need for compartmentalization or immobilization of multiplereaction components (e.g., both primers) during amplification. Instead,mixtures of polynucleotide templates in solution can be directlycontacted with amplification reaction components and a suitable surfaceor support having a first primer attached thereto. Other componentsrequired for amplification can be provided in the same continuous liquidphase, including a polymerase, one or more types of nucleotide andoptionally a second primer. In some embodiments, the reaction mixturealso includes a recombinase. Optionally, the reaction mixture furtherincludes at least one agent selected from the group consisting of: adiffusion limiting agent, a sieving agent, and a crowding agent.Examples of amplification mixtures suitable for achieving monoclonalamplification of templates contained in a single continuous liquid phaseare described further herein. Optionally, different templates can beamplified onto different locations on a single surface or support, ordifferent templates can be amplified onto different surfaces ordifferent supports within the same reaction mixture.

In some embodiments, the methods for nucleic acid amplification includemixing one or more nucleic acid templates with one or more primers inthe presence of one of more enzymes capable of polymerization. In someembodiments, the one or more enzymes capable of polymerization includeat least one polymerase and a recombinase. In some embodiments, the atleast one polymerase includes a thermostable or thermolabile polymerase.In some embodiments, the at least one polymerase includes a biologicallyactive fragment of a DNA or RNA polymerase that maintains sufficientcatalytic activity to polymerize or incorporate at least one nucleotideunder any suitable conditions. In one embodiment, the at least onepolymerase comprises a mutated DNA or RNA polymerase that maintainssufficient catalytic activity to perform nucleotide polymerization underany suitable conditions. In another embodiment, the at least onepolymerase includes one or more amino acid mutations that do not disruptprocessivity of the polymerase; and wherein the at least one polymerasehaving at least one mutation maintains sufficient catalytic activity toperform polymerization.

In some embodiments, the methods for nucleic acid amplification includemixing one or more nucleic acid templates with one or more primers inthe presence of one of more enzymes capable of polymerization. In someembodiments, the at least one polymerase is an A family DNA polymerasefrom selected from the group consisting of a Pol I-type DNA polymerasesuch as E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, Omni Klen Taq DNA polymerase series, Klen Taq DNApolymerase series, T7 DNA polymerase, T5 DNA polymerase, T4 DNApolymerase, and Tth DNA polymerase. In another embodiment, the one ormore of the enzymes capable of polymerization can include any one ormore of the following B family DNA polymerases: Bst polymerase, Tlipolymerase, Pfu polymerase, Pfu turbo polymerase, Pyrobest polymerase,Pwo polymerase, KOD polymerase, Sac polymerase, Sau polymerase, Ssopolymerase, Poc polymerase, Pab polymerase, Mth polymerase, Phopolymerase, ES4 polymerase, VENT polymerase, DEEPVENT polymerase,Therminator™ polymerase, phage Phi29 polymerase, and phage B103polymerase. In another embodiment, the methods for nucleic acidamplification can include one or more reverse transcriptase. This isparticularly useful when the nucleic acid template is mRNA.

In yet another embodiment, the one or more enzymes capable ofpolymerization can include any suitable bacterial DNA polymeraseincluding without limitation E. coli DNA polymerases I, II and III, IVand V, the Klenow fragment of E. coli DNA polymerase, Bacillusstearothermophilus (Bst) DNA polymerase, Staphylococcus aureus (Sau) DNApolymerase and Sulfolobus solfataricus (Sso) DNA polymerase.

In some embodiments, the one or more enzymes capable of polymerizationcan include any suitable viral and/or phage DNA polymerase includingwithout limitation T4 DNA polymerase, T5 DNA polymerase (see, e.g., U.S.Pat. No. 5,716,819), T7 DNA polymerase, Phi-15 DNA polymerase, Phi-29DNA polymerase (see, e.g., U.S. Pat. No. 5,198,543; also referred tovariously as Φ29 polymerase, phi29 polymerase, phi 29 polymerase, Phi 29polymerase, and Phi29 polymerase); Φ15 polymerase (also referred toherein as Phi-15 polymerase); Φ21 polymerase (Phi-21 polymerase); PZApolymerase; PZE polymerase, PRD1 polymerase; Nf polymerase; M2Ypolymerase; SF5 polymerase; f1 DNA polymerase, Cp-1 polymerase; Cp-5polymerase; Cp-7 polymerase; PR4 polymerase; PR5 polymerase; PR722polymerase; L17 polymerase; M13 DNA polymerase, RB69 DNA polymerase, G1polymerase; GA-1 polymerase, BS32 polymerase; B103 polymerase; apolymerase obtained from any phi-29 like phage or derivatives thereof,etc. See, e.g., U.S. Pat. No. 5,576,204, filed Feb. 11, 1993; U.S. Pat.Appl. No. 2007/0196846, published Aug. 23, 2007.

In one embodiment, the one or more enzymes capable of polymerizationinclude a T5 or T7 DNA polymerase. In some embodiments, the one or moreenzymes capable of polymerization include a T5 or T7 DNA polymerasehaving one or more amino acid mutations that reduce 3′-5′ exonucleaseactivity. In some embodiments, the T5 or T7 DNA polymerase having one ormore amino acid mutations that reduce 3′-5′ exonuclease activity, doesnot contain an amino acid mutation that disrupts processivity of the T5or T7 DNA polymerase. In some embodiments, the T5 or T7 DNA polymerasecan include one or more amino acid mutations that eliminate detectable3′-5′ exonuclease activity; and wherein the one or more amino acidmutations do not disrupt processivity of the T5 or T7 DNA polymerase.

In yet another embodiment, the one or more enzymes capable ofpolymerization can include any suitable archaeal DNA polymeraseincluding without limitation the thermostable and/or thermophilic DNApolymerases such as, for example, DNA polymerases isolated from Thermusaquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase,Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth)DNA polymerase, Thermus flavus (Tfl) DNA polymerase, Pyrococcus woesei(Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase as wellas Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymeraseor Vent DNA polymerase, Pyrococcus sp. GB-D polymerase, “Deep Vent” DNApolymerase, New England Biolabs), Thermotoga maritima (Tma) DNApolymerase, Bacillus stearothermophilus (Bst) DNA polymerase, PyrococcusKodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp.JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNApolymerase, Thermococcus acidophilium DNA polymerase; Sulfolobusacidocaldarius DNA polymerase; Thermococcus sp. 9° N-7 DNA polymerase;Thermococcus sp. NA1; Pyrodictium occultum DNA polymerase; Methanococcusvoltae DNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;the heterodimeric DNA polymerase DP1/DP2, etc.

In some embodiments, the one or more enzymes capable of polymerizationcan include any suitable RNA polymerase. Suitable RNA polymerasesinclude, without limitation, T3, T5, T7, and SP6 RNA polymerases.

In some embodiments, the disclosure relates generally to compositionscomprising nucleic acids produced by any of the methods describedherein. In some embodiments, the disclosure generally relates tocompositions for nucleic acid amplification. In some embodiments, thecomposition for nucleic acid amplification includes at least onepolymerase and a recombinase. In some embodiments, the compositionincludes a bacteriophage polymerase comprising a T5 DNA polymerase or T7DNA polymerase. In some embodiments, the composition for nucleic acidamplification may further include one or more co-factors or accessoryproteins. In one embodiment, the composition includes a T7 DNApolymerase and one or more co-factors, such as thioredoxin. In someembodiments, the T7 DNA polymerase and thioredoxin co-factor can beexpressed independently from distinct expression cassettes or expressionvectors prior to use in the nucleic acid amplification method. Inanother embodiment, the T7 DNA polymerase and thioredoxin co-factor canbe co-expressed from an expression cassette or expression vector priorto use in the nucleic acid amplification methods disclosed herein (Seefor example, U.S. Pat. No. 4,795,699). Expression cassettes andexpression vectors are well known in the art. Additionally, methods ofexpressing bacteriophage polymerases such as T5 and T7 polymerases arealso well known in the art; see for example U.S. Pat. No. 5,716,819. Insome embodiments, a composition for nucleic acid amplification comprisesa T7 DNA polymerase, thioredoxin and a recombinase. In some embodiments,the composition for nucleic acid amplification can comprise a T5 DNApolymerase and a recombinase. In some embodiments, the recombinasecomprises UvsX, RecA, or functional analogs thereof. In someembodiments, the recombinase can include additional components thatenhance or facilitate recombinase polymerase amplification. In someembodiments, the additional components that enhance or facilitaterecombinase polymerase amplification include one or more accessoryproteins and/or single-stranded binding proteins. In some embodiments,the one or more accessory proteins can include UvsY; and the one or moresingle-stranded proteins can include gp32. Optionally, a crowding agent,gelling agent, hydrogel, sieving agent, or other suitable recombinasepolymerase amplification reagents can be included as components. Someexamples or recombinase polymerase amplification components aredescribed in U.S. Pat. No. 8,062,850, which is hereby incorporated byreference in its entirety.

In some embodiments, the one or more enzymes capable of polymerizationcan include at least one polymerase derived from bacteria (includingmutant, derivative, synthetic or engineered forms of a naturallyoccurring bacterial polymerase). In one embodiment, the at least onepolymerase derived from bacteria can include a Staphylococcus aureuspolymerase. In another embodiment, the at least one polymerase derivedfrom bacteria can include Sau polymerase (Staphylococcus aureus);optionally in the presence of one or more additional DNA polymerases. Insome embodiments, the composition for nucleic acid amplification caninclude a Sau polymerase and a bacteriophage polymerase. In someembodiments, the composition for nucleic acid amplification can includea Sau polymerase and a T7 DNA polymerase; optionally the compositionfurther comprises a recombinase, and one or more accessory proteinsand/or single-stranded binding proteins. In some embodiments,compositions for nucleic acid amplification comprise one or moreaccessory proteins. For example, an accessory protein can improve theactivity of a recombinase enzyme (U.S. Pat. No. 8,071,308 granted toPiepenburg, et al.). In some embodiments, an accessory protein can bindsingle strands of nucleic acids, or can load a recombinase onto anucleic acid. In some embodiments, an accessory protein compriseswild-type, mutant, recombinant, fusion, or fragments thereof. In someembodiments, accessory proteins can originate from any combination ofthe same or different species as the recombinase enzyme that are used toconduct a nucleic acid amplification reaction. Accessory proteins canoriginate from any bacteriophage including a myoviral phage. In someembodiments, compositions for nucleic acid amplification can includesingle-stranded binding proteins. Single-stranded binding proteinsinclude myoviral gp32 (e.g., T4 or RB69), Sso SSB from Sulfolobussolfataricus, MjA SSB from Methanococcus jannaschii, or E. coli SSBprotein.

In another embodiment, the disclosure generally relates to a compositionfor nucleic acid amplification; wherein the composition includes atleast one polymerase, a recombinase and at least one accessory protein.In some embodiments, the at least one polymerase includes a Saupolymerase in combination or admixed with a T7 DNA polymerase; therecombinase, UvsX; and the accessory protein, UvsY. Optionally, thecomposition further includes thioredoxin and single-stranded bindingprotein, gp32.

In another embodiment, the composition for nucleic acid amplificationincludes a Sau polymerase, a T7 DNA polymerase, a recombinase andthioredoxin. In yet another embodiment, the composition for nucleic acidamplification includes a T7 DNA polymerase, a recombinase, UvsY, gp32and thioredoxin. In yet another embodiment, the composition for nucleicacid amplification includes a Sau polymerase, a T5 DNA polymerase, UvsX,UvsY and gp32.

In one embodiment, the composition comprising a T7 DNA polymerase mayoptionally include a T7 DNA polymerase having reduced 3′-5′ exonucleaseactivity. In another embodiment, the T7 DNA polymerase includes a T7 DNApolymerase lacking detectable 3′-5′ exonuclease activity.

In some embodiments, the nucleic acid amplification reactions disclosedherein include one or more enzymes capable of polymerization; whereinthe one or more enzymes capable of polymerization may be blended, mixed,or present within the same amplification reaction mixture. In someembodiments, the presence of one or more enzymes capable ofpolymerization co-existing in the same nucleic acid amplificationreaction mixture can provide superior nucleic acid synthesis as comparedto an amplification reaction mixture containing a single polymerizingenzyme. In some embodiments, superior nucleic acid synthesis can bedetermined by measuring one or more downstream sequencing metrics knownto one of ordinary skill in the art. In one embodiment, the one or moresequencing metrics may optionally include total yield of synthesizedproduct, raw accuracy, template dissociation, total sequence throughput(e.g., throughput of a single sequencing run), template affinity, AQ17,AQ20 or read length.

In some embodiments, the nucleic acid amplification compositionsdisclosed herein include one or more enzymes capable of polymerization.In some embodiments, the nucleic acid amplification compositions includeone or more polymerases; optionally having one or more site-specificamino acid mutations that modulate performance of one or more sequencingmetrics known to one of ordinary skill in the art. In some embodiments,the one or more polymerases can include one or more site-specific aminoacid mutations that modulate performance (relative to the correspondingwild type or unmutated polymerase) as measured by any one or moresequencing metrics selected from the group consisting of read length,template dissociation, raw accuracy, template affinity, throughput,AQ17, AQ20 and signal to noise ratio.

In some embodiments, the one or more polymerases having one or moresite-specific amino acid mutations can exhibit increased or superiorperformance relative to the corresponding unmutated (e.g., wild-type)polymerase. Optionally, the increased or superior performance ismeasured using one or more sequencing metrics selected from the groupconsisting of read length, template dissociation, raw accuracy, templateaffinity, total sequencing throughput (e.g., throughput per sequencingrun), AQ17, AQ20 and signal to noise ratio. In another embodiment, oneor more polymerases having one or more site-specific amino acidmutations can decrease performance of one or more sequencing metricsselected from the group consisting of read length, templatedissociation, raw accuracy, template affinity, total sequencingthroughput (e.g., throughput per sequencing run), AQ17, AQ20 and signalto noise ratio.

In some embodiments, the nucleic acid amplification compositionsdisclosed herein include one or more enzymes capable of catalyzingnucleotide incorporation. In some embodiments, the nucleic acidamplification compositions capable of catalyzing nucleotideincorporation include one or more polymerases; optionally having one ormore site-specific amino acid mutations that modulate performance(relative to the corresponding wild type or unmutated polymerase)according to one or more sequencing metrics known to one of ordinaryskill in the art. In some embodiments, the one or more polymerasescapable of catalyzing nucleotide incorporation include one or moresite-specific amino acid mutations that modulate performance (relativeto the corresponding wild type or unmutated polymerase) according to oneor more sequencing metrics selected from the group consisting of readlength, template dissociation, raw accuracy, template affinity,throughput, AQ17, AQ20 and signal to noise ratio. In some embodiments,the one or more polymerases capable of catalyzing nucleotideincorporation include one or more site-specific amino acid mutationsthat can increase performance (relative to the corresponding wild typeor unmutated polymerase) according to one or more sequencing metricsselected from the group consisting of read length, templatedissociation, raw accuracy, template affinity, throughput, AQ17, AQ20and signal to noise ratio. In another embodiment, one or morepolymerases capable of catalyzing nucleotide incorporation include oneor more site-specific amino acid mutations that decrease performance(relative to the corresponding wild type or unmutated polymerase)according to one or more sequencing metrics selected from the groupconsisting of read length, template dissociation, raw accuracy, templateaffinity, throughput, AQ17, AQ20 and signal to noise ratio.

In some embodiments, the nucleic acid amplification compositiondisclosed herein includes one or more enzymes capable of polymerization,wherein the one or more enzymes include one or more DNA or RNApolymerases. In some embodiments, the one or more DNA or RNA polymerasesinclude at least one polymerase having reduced 3′-5′ exonucleaseactivity (relative to the corresponding wild type polymerase). In someembodiments, at least one of the one or more polymerases lacksdetectable 3′-5′ exonuclease activity. In some embodiments, the at leastone of the one or more polymerases includes one or more amino acidmutations that results in a polymerase lacking detectable 3′-5′exonuclease activity and wherein the one or more amino acid mutationsdoes not disrupt or significantly the polymerase processivity (relativeto the corresponding wild type or unmutated polymerase).

In some embodiments, the disclosure relates generally to compositionsfor nucleic acid amplification comprising a DNA polymerase havingreduced 3′-5′ exonuclease activity (relative to the corresponding wildtype polymerase). The 3′-5′ exonuclease activity of DNA polymerases isoften undesirable in sequencing environments. Therefore, the disclosuregenerally relates to a composition comprising a DNA polymerasepossessing reduced 3′-5′ exonuclease activity or a compositioncomprising a DNA polymerase that lacks detectable 3′-5′ exonucleaseactivity. 3′-5′ exonuclease activity is often associated with DNApolymerases and is generally considered to be involved in DNAreplication. As used herein, polymerases having reduced 3′-5′exonuclease activity include any polymerase having less than about 90%,80%, 70%, 60%, 50%, 40%, 30%, 20%, or 15%, 3′-5′ exonuclease activity ascompared to a corresponding wild-type polymerase having 3′-5′exonuclease activity. As used herein, a polymerase lacking detectable3′-5′ exonuclease activity, refers to polymerase with less than 10%, 5%,3%, 2% or 1%, 3′-5′ exonuclease activity as compared to a correspondingwild-type polymerase having 3′-5′ exonuclease activity.

In some embodiments, the composition comprises a recombinase and a DNApolymerase, wherein the DNA polymerase possesses reduced 3′-5′exonuclease activity as compared to a corresponding wild-type DNApolymerase. In some embodiments, the composition comprises a recombinaseand a DNA polymerase, wherein the DNA polymerase lacks detectable 3′-5′exonuclease activity as compared to a corresponding wild-type DNApolymerase. In some embodiments, the recombinase comprises RecA or UvsXor a biologically active fragment thereof. In some embodiments, therecombinase further includes one or more accessory proteins and/or oneor more single-stranded binding proteins. In one embodiment, the one ormore accessory proteins include UvsY. In some embodiments, the one ormore single-stranded proteins include gp32. In some embodiments, thecomposition further includes a Sau polymerase.

In some embodiments, the disclosure relates to a composition for nucleicacid amplification comprising a recombinase and a T7 DNA polymerasehaving reduced 3′-5′ exonuclease activity. In some embodiments, the T7DNA polymerase lacks detectable 3′-5′ exonuclease activity. In someembodiments, the composition comprises a T7 DNA polymerase having one ormore site-specific amino acid mutations that results in reduced 3′-5′exonuclease activity (See, for example, J. Biol Chem, 1997,272:6599-6606). In some embodiments, the composition comprises a T7 DNApolymerase having one or more amino acid mutations that results in a T7DNA polymerase lacking detectable 3′-5′ exonuclease activity. In someembodiments, the T7 polymerase includes bacteriophage T7 DNA polymerasehaving an amino acid mutation at the 5^(th) amino acid residue (SeePatel et al., Biochemistry, 1991, 30:511-525). In some embodiments, theDNA polymerase having reduced 3′-5′ exonuclease activity includes one ormore amino acid substitutions. The one or more amino acid substitutionscan optionally occur at positions 5 or 7, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 1. In someembodiments, the DNA polymerase having reduced 3′-5′ exonucleaseactivity optionally includes one, two, three, four, five, or moreadditional amino acid substitutions, wherein the additional amino acidsubstitutions do not disrupt processivity of the DNA polymerase. In someembodiments, the DNA polymerase lacking detectable 3′-5′ exonucleaseactivity includes one or more amino acid substitutions. The one or moreamino acid substitutions can optionally occur at positions 5 and 7,wherein the numbering is relative to the amino acid sequence of SEQ IDNO: 1. In some embodiments, the DNA polymerase lacking detectable 3′-5′exonuclease activity optionally includes one, two, three, four, five, ormore additional amino acid substitutions, wherein the additional aminoacid substitutions do not disrupt processivity of the DNA polymerase. Insome embodiments, the T7 DNA polymerase includes a D5A mutation (SEQ IDNO. 2). In some embodiments, the bacteriophage T7 polymerase includes anamino acid mutation at the 7^(th) amino acid residue (See Patel et al.,Biochemistry, 1991, 30:511-525). In some embodiments, the bacteriophageT7 DNA polymerase includes an E7A mutation (SEQ ID NO. 3). In someembodiments, the T7 DNA polymerase includes a D5A amino acid mutationand an E7A mutation (See Patel et al., Biochemistry, 1991, 30:511-525)(SEQ ID NO. 4). In some embodiments, the T7 DNA polymerase includes anaspartic acid to alanine mutation and optionally a glutamic acid toalanine mutation. In some embodiments, the disclosure relates generallyto a nucleic acid amplification composition comprising a recombinase, asingle-strand binding protein, one or more accessory proteins,thioredoxin, a Sau polymerase and a T7 DNA polymerase, wherein the T7DNA polymerase includes an amino acid mutation that does not disruptprocessivity of the T7 DNA polymerase. In some embodiments, the aminoacid mutation of the T7 DNA polymerase includes D5A and/or E7A. In someembodiments, the composition comprising a T7 DNA polymerase with anamino acid mutation at D5A and/or E7A further includes an amino acidmutation that does not disrupt processivity or diminishes catalyticactivity. Several recombinant 3′-5′ exonuclease minus polymerases(polymerases exhibiting no detectable degradation of single or doublestranded templates) are known in the art. For example, see DNApolymerase I Klenow fragment, exo-available from US Biological (MA) thatlacks both 3′-5′ and 5′-3′ exonuclease activity and Vent^(R) (exo-) DNApolymerase available from New England Biolabs (MA).

In some embodiments, the composition for nucleic acid amplificationcomprises a recombinase and a T7 DNA polymerase, wherein the T7 DNApolymerase possesses reduced 3′-5′ exonuclease activity as compared to acorresponding wild-type T7 DNA polymerase.

In some embodiments, the composition comprises a recombinase and a T7DNA polymerase, wherein the T7 DNA polymerase lacks detectable 3′-5′exonuclease activity as compared to a corresponding wild-type T7 DNApolymerase. In some embodiments, the recombinase comprises RecA or UvsXor a biologically active fragment thereof. In some embodiments, therecombinase further includes one or more accessory proteins and/or oneor more single-stranded binding proteins. In one embodiment, the one ormore accessory proteins include UvsY. In some embodiments, the one ormore single-stranded proteins include gp32. In some embodiments, thecomposition further includes a Sau polymerase. In another embodiment,the composition further includes thioredoxin.

In some embodiments, the disclosure relates generally to a compositioncomprising a DNA polymerase having reduced 3′-5′ exonuclease activity.In some embodiments, the composition comprises a T5 DNA polymerasehaving reduced 3′-5′ exonuclease activity. In contrast to bacteriophageT7 DNA polymerase, bacteriophage T5 DNA polymerase does not require aco-factor. Therefore, in some embodiments, a composition for nucleicacid amplification comprises a bacteriophage T5 DNA polymerase havingreduced 3′-5′ exonuclease activity. In some embodiments, the T5 DNApolymerase includes a polymerase lacking detectable 3′-5′ exonucleaseactivity. In some embodiments, the composition for nucleic acidamplification comprises a T5 DNA polymerase having one or more aminoacid mutations that results in a T5 DNA polymerase lacking detectable3′-5′ exonuclease activity (See U.S. Pat. No. 5,716,819). In someembodiments, the bacteriophage T5 DNA polymerase includes an Asp-138deletion or substitution (See U.S. Pat. No. 5,716,819, herebyincorporated by reference in its entirety for all purposes). In someembodiments, the bacteriophage T5 DNA polymerase includes an amino acidmutation at the 138^(th) amino acid residue of the amino acid sequencedisclosed in U.S. Pat. No. 5,716,819. In some embodiments, thebacteriophage T5 DNA polymerase includes an Asp to Ala mutation. In someembodiments, the bacteriophage T5 DNA polymerase includes a Glu¹⁴⁰deletion or substitution (See U.S. Pat. No. 5,716,819). In someembodiments, the bacteriophage T5 polymerase includes an amino acidmutation at the 140^(th) amino acid residue (See U.S. Pat. No.5,716,819). In some embodiments, the bacteriophage T5 DNA polymeraseincludes a Glu to Ala mutation. In some embodiments, the bacteriophageT5 DNA polymerase includes an Asp¹³⁸ to Ala¹³⁸ amino acid mutation and aGlu¹⁴⁰ to Ala¹⁴⁰ mutation (See U.S. Pat. No. 5,716,819). In someembodiments, the disclosure relates generally to a nucleic acidamplification composition comprising a recombinase and a T5 DNApolymerase, wherein the T5 DNA polymerase includes an amino acidmutation resulting in reduced 3′-5′ exonuclease activity as compared towild-type T5 DNA polymerase. In some embodiments, the disclosure relatesgenerally to a nucleic acid amplification composition comprising arecombinase and a T5 DNA polymerase, wherein the T5 DNA polymeraseincludes an amino acid mutation resulting in no detectable 3′-5′exonuclease activity as compared to wild-type T5 DNA polymerase.

In some embodiments, the composition for nucleic acid amplificationcomprises a recombinase and a T5 DNA polymerase, wherein the T5 DNApolymerase possesses reduced 3′-5′ exonuclease activity as compared to acorresponding wild-type T5 DNA polymerase.

In some embodiments, the composition comprises a recombinase and a T5DNA polymerase, wherein the T5 DNA polymerase lacks detectable 3′-5′exonuclease activity as compared to a corresponding wild-type T5 DNApolymerase. In some embodiments, the recombinase comprises RecA or UvsXor a biologically active fragment thereof. In some embodiments, therecombinase further includes one or more accessory proteins and/or oneor more single-stranded binding proteins. In one embodiment, the one ormore accessory proteins include UvsY. In some embodiments, the one ormore single-stranded proteins include gp32. In some embodiments, thecomposition further includes a Sau polymerase.

In some embodiments, the nucleic acid amplification compositioncomprises a recombinase and at least one polymerase having one, two,three, four or more amino acid mutations that increase polymeraseperformance (relative to the corresponding wild type or unmutatedpolymerase) as measured by any one or more sequencing metrics selectedfrom the group consisting of: read length, template dissociation, rawaccuracy, affinity, throughput, AQ17, AQ20, and signal to noise ratio.In some embodiments, the at least one polymerase having one, two, three,four or more, amino acid mutations can optionally include a frameshiftmutation. In some embodiments, the at least one polymerase can includeone, two, three, four or more site-specific DNA mutations that increaseperformance (relative to the corresponding wild type or unmutatedpolymerase) according to any one or more sequencing metrics selectedfrom the group consisting of read length, template dissociation, rawaccuracy, affinity, throughput, AQ17, AQ20, and signal to noise ratio.

In some embodiments, a polymerase for use in the nucleic acid synthesisand nucleic acid amplification methods disclosed herein can include amutant, engineered, derivative or variant DNA polymerase with enhancedprocessivity as compared to a corresponding wild-type DNA polymerase. Insome embodiments, a blend or mixture of polymerases can include one ormore DNA polymerases with enhanced processivity as compared toprocessivity of a blend or mixture of corresponding wild-type DNApolymerases. By processivity, it is meant that the DNA polymerase isable to continuously incorporate multiple nucleotides using the sameprimer-nucleic acid template without dissociating from the nucleic acidtemplate. In some embodiments, the processivity of a given polymerasecan be measured in terms of the average number of nucleotidesincorporated by the polymerase prior to dissociating from the templateunder a defined set of reactions. In some embodiments, a polymerase foruse in the nucleic acid amplification methods disclosed herein caninclude a DNA polymerase or blend of DNA polymerases having aprocessivity of 100, 200, 300, 400, 500, 600 base pairs, or longer. Insome embodiments, a polymerase for use in the nucleic acid amplificationmethods disclosed herein can include a DNA polymerase with enhancedprocessivity of at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or greater,processivity as compared to a corresponding wild-type DNA polymerase. Insome embodiments, a blend of DNA polymerases with enhanced processivityfor use in the nucleic acid amplification methods disclosed herein caninclude a processivity of at least 10%, 20%, 25%, 30%, 35%, 40%, 45% orgreater, processivity as compared to a blend of corresponding wild-typeDNA polymerases. Processivity of polymerases is well known in the art,and can be readily determined via various kinetic experiments, forexample, see Cannistraro and Taylor, J. Biol. Chem (2004)18:18288-18295.

In some embodiments, the disclosure relates generally to a compositionfor nucleic acid synthesis or nucleic acid amplification comprising arecombinase and at least one DNA polymerase. In some embodiments, the atleast one polymerase includes a DNA polymerase with enhanced read-lengthcapability as compared to a corresponding wild-type DNA polymerase. Insome embodiments, the at least one polymerase includes a blend ormixture of DNA polymerases with enhanced read-length capability ascompared to read-length capability of a corresponding blend of wild-typeDNA polymerases. By enhanced read-length capability, it is meant thatthe at least one DNA polymerase (or blend of DNA polymerases) is able tocontinuously incorporate multiple nucleotides using the sameprimer-nucleic acid template without dissociating from the template andthat the length of the resulting extended primer product (generatedduring amplification) is longer (as measured by nucleotide extension),than the length of an extended primer product generated under comparableconditions, using a corresponding wild type DNA polymerase (or blend ofwild-type DNA polymerases). In some embodiments, a DNA polymerase havingenhanced read-length capability comprises a read length that is at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or greater, in readlength than the read-length obtained under comparable conditions using acorresponding wild type DNA polymerase.

SEQ ID NO: 1 MIVSDIEANALLESVTKFHCGVIYDYSTAEYVSYRPSDFGAYLDALEAEVARGGLIVFHNGHKYDVPALTKLAKLQLNREFHLPRENCIDTLVLSRLIHSNLKDTDMGLLRSGKLPGKRFGSHALEAWGYRLGEMKGEYKDDFKRMLEEQGEEYVDGMEWWNFNEEMMDYNVQDVVVTKALLEKLLSDKHYFPPEIDFTDVGYTTFWSESLEAVDIEHRAAWLLAKQERNGFPFDTKAIEELYVELAARRSELLRKLTETFGSWYQPKGGTEMFCHPRTGKPLPKYPRIKTPKVGGIFKKPKNKAQREGREPCELDTREYVAGAPYTPVEHVVFNPSSRDHIQKKLQEAGWVPTKYTDKGAPVVDDEVLEGVRVDDPEKQAAIDLIKEYLMIQKRIGQSAEGDKAWLRYVAEDGKIHGSVNPNGAVTGRATHAFPNLAQIPGVRSPYGEQCRAAFGAEHHLDGITGKPWVQAGIDASGLELRCLAHFMARFDNGEYAHEILNGDIHTKNQIAAELPTRDNAKTFIYGFLYGAGDEKIGQIVGAGKERGKELKKKFLENTPAIAALRESIQQTLVESSQWVAGEQQVKWKRRWIKGLDGRKVHVRSPHAALNTLLQSAGALICKLWIIKTEEMLVEKGLKHGWDGDFAYMAWVHDEIQVGCRTEEIAQVVIETAQEAMRWVGDHWNF RCLLDTEGKMGPNWAICH.SEQ ID NO: 2 MIVSAIEANALLESVTKFHCGVIYDYSTAEYVSYRPSDFGAYLDALEAEVARGGLIVFHNGHKYDVPALTKLAKLQLNREFHLPRENCIDTLVLSRLIHSNLKDTDMGLLRSGKLPGKRFGSHALEAWGYRLGEMKGEYKDDFKRMLEEQGEEYVDGMEWWNFNEEMMDYNVQDVVVTKALLEKLLSDKHYFPPEIDFTDVGYTTFWSESLEAVDIEHRAAWLLAKQERNGFPFDTKAIEELYVELAARRSELLRKLTETFGSWYQPKGGTEMFCHPRTGKPLPKYPRIKTPKVGGIFKKPKNKAQREGREPCELDTREYVAGAPYTPVEHVVFNPSSRDHIQKKLQEAGWVPTKYTDKGAPVVDDEVLEGVRVDDPEKQAAIDLIKEYLMIQKRIGQSAEGDKAWLRYVAEDGKIHGSVNPNGAVTGRATHAFPNLAQIPGVRSPYGEQCRAAFGAEHHLDGITGKPWVQAGIDASGLELRCLAHFMARFDNGEYAHEILNGDIHTKNQIAAELPTRDNAKTFIYGFLYGAGDEKIGQIVGAGKERGKELKKKFLENTPAIAALRESIQQTLVESSQWVAGEQQVKWKRRWIKGLDGRKVHVRSPHAALNTLLQSAGALICKLWIIKTEEMLVEKGLKHGWDGDFAYMAWVHDEIQVGCRTEEIAQVVIETAQEAMRWVGDHWNF RCLLDTEGKMGPNWAICH.SEQ ID NO: 3 MIVSDIAANALLESVTKFHCGVIYDYSTAEYVSYRPSDFGAYLDALEAEVARGGLIVFHNGHKYDVPALTKLAKLQLNREFHLPRENCIDTLVLSRLIHSNLKDTDMGLLRSGKLPGKRFGSHALEAWGYRLGEMKGEYKDDFKRMLEEQGEEYVDGMEWWNFNEEMMDYNVQDVVVTKALLEKLLSDKHYEPPEIDETDVGYTTEWSESLEAVDIEHRAAWLLAKQERNGFPFDTKAIEELYVELAARRSELLRKLTETFGSWYQPKGGTEMFCHPRTGKPLPKYPRIKTPKVGGIFKKPKNKAQREGREPCELDTREYVAGAPYTPVEHVVFNPSSRDHIQKKLQEAGWVPTKYTDKGAPVVDDEVLEGVRVDDPEKQAAIDLIKEYLMIQKRIGQSAEGDKAWLRYVAEDGKIHGSVNPNGAVTGRATHAFPNLAQIPGVRSPYGEQCRAAFGAEHHLDGITGKPWVQAGIDASGLELRCLAHFMARFDNGEYAHEILNGDIHTKNQIAAELPTRDNAKTFIYGFLYGAGDEKIGQIVGAGKERGKELKKKFLENTPAIAALRESIQQTLVESSQWVAGEQQVKWKRRWIKGLDGRKVHVRSPHAALNTLLQSAGALICKLWIIKTEEMLVEKGLKHGWDGDFAYMAWVHDEIQVGCRTEEIAQVVIETAQEAMRWVGDHWNF RCLLDTEGKMGPNWAICH.SEQ ID NO: 4 MIVSAIAANALLESVTKFHCGVIYDYSTAEYVSYRPSDFGAYLDALEAEVARGGLIVFHNGHKYDVPALTKLAKLQLNREFHLPRENCIDTLVLSRLIHSNLKDTDMGLLRSGKLPGKRFGSHALEAWGYRLGEMKGEYKDDFKRMLEEQGEEYVDGMEWWNFNEEMMDYNVQDVVVTKALLEKLLSDKHYFPPEIDFTDVGYTTFWSESLEAVDIEHRAAWLLAKQERNGFPFDTKAIEELYVELAARRSELLRKLTETFGSWYQPKGGTEMFCHPRTGKPLPKYPRIKTPKVGGIFKKPKNKAQREGREPCELDTREYVAGAPYTPVEHVVFNPSSRDHIQKKLQEAGWVPTKYTDKGAPVVDDEVLEGVRVDDPEKQAAIDLIKEYLMIQKRIGQSAEGDKAWLRYVAEDGKIHGSVNPNGAVTGRATHAFPNLAQIPGVRSPYGEQCRAAFGAEHHLDGITGKPWVQAGIDASGLELRCLAHFMARFDNGEYAHEILNGDIHTKNQIAAELPTRDNAKTFIYGFLYGAGDEKIGQIVGAGKERGKELKKKFLENTPAIAALRESIQQTLVESSQWVAGEQQVKWKRRWIKGLDGRKVHVRSPHAALNTLLQSAGALICKLWIIKTEEMLVEKGLKHGWDGDFAYMAWVHDEIQVGCRTEEIAQVVIETAQEAMRWVGDHWNF RCLLDTEGKMGPNWAICH.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control.

It will be appreciated that there is an implied “about” prior to thetemperatures, concentrations, times, etc., discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”,“containing”, “include”, “includes”, and “including” are not intended tobe limiting.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention.

Unless otherwise defined, scientific and technical terms used inconnection with the present teachings described herein shall have themeanings that are commonly understood by those of ordinary skill in theart. Generally, nomenclatures utilized in connection with, andtechniques of, cell and tissue culture, molecular biology, and proteinand oligo- or polynucleotide chemistry and hybridization describedherein are those well known and commonly used in the art. Standardtechniques are used, for example, for nucleic acid purification andpreparation, chemical analysis, recombinant nucleic acid, andoligonucleotide synthesis. Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications oras commonly accomplished in the art or as described herein. Thetechniques and procedures described herein are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the instant specification. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (Third ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques described herein are those well known and commonly usedin the art.

As utilized in accordance with exemplary embodiments provided herein,the following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein the term “amplification” and its variants includes anyprocess for producing multiple copies or complements of at least someportion of a polynucleotide, said polynucleotide typically beingreferred to as a “template”. The template polynucleotide can be singlestranded or double stranded. Amplification of a given template canresult in the generation of a population of polynucleotide amplificationproducts, collectively referred to as an “amplicon”. The polynucleotidesof the amplicon can be single stranded or double stranded, or a mixtureof both. Typically, the template will include a target sequence, and theresulting amplicon will include polynucleotides having a sequence thatis either substantially identical or substantially complementary to thetarget sequence. In some embodiments, the polynucleotides of aparticular amplicon are substantially identical, or substantiallycomplementary, to each other; alternatively, in some embodiments thepolynucleotides within a given amplicon can have nucleotide sequencesthat vary from each other. Amplification can proceed in linear orexponential fashion, and can involve repeated and consecutivereplications of a given template to form two or more amplificationproducts. Some typical amplification reactions involve successive andrepeated cycles of template-based nucleic acid synthesis, resulting inthe formation of a plurality of daughter polynucleotides containing atleast some portion of the nucleotide sequence of the template andsharing at least some degree of nucleotide sequence identity (orcomplementarity) with the template. In some embodiments, each instanceof nucleic acid synthesis, which can be referred to as a “cycle” ofamplification, includes primer annealing and primer extension steps;optionally, an additional denaturation step can also be included whereinthe template is partially or completely denatured. In some embodiments,one round of amplification includes a given number of repetitions of asingle cycle of amplification. For example, a round of amplification caninclude 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or more repetitionsof a particular cycle. In one exemplary embodiment, amplificationincludes any reaction wherein a particular polynucleotide template issubjected to two consecutive cycles of nucleic acid synthesis. Thesynthesis can include template-dependent nucleic acid synthesis. Eachcycle of nucleic acid synthesis optionally includes a single primerannealing step and a single extension step. In some embodiments,amplification includes isothermal amplification.

As used herein, the term “contacting” and its variants, when used inreference to any set of components, includes any process whereby thecomponents to be contacted are mixed into same mixture (for example, areadded into the same compartment or solution), and does not necessarilyrequire actual physical contact between the recited components. Therecited components can be contacted in any order or any combination (orsubcombination), and can include situations where one or some of therecited components are subsequently removed from the mixture, optionallyprior to addition of other recited components. For example, “contactingA with B and C” includes any and all of the following situations: (i) Ais mixed with C, then B is added to the mixture; (ii) A and B are mixedinto a mixture; B is removed from the mixture, and then C is added tothe mixture; and (iii) A is added to a mixture of B and C. “Contacting atemplate with a reaction mixture” includes any or all of the followingsituations: (i) the template is contacted with a first component of thereaction mixture to create a mixture; then other components of thereaction mixture are added in any order or combination to the mixture;and (ii) the reaction mixture is fully formed prior to mixture with thetemplate.

As used herein, the term “support” and its variants include any solid orsemisolid article on which reagents such as nucleic acids can beimmobilized.

As used herein, the term “isothermal” and its variants, when used inreference to reference to any reaction conditions, process or method,includes conditions, processes and methods that are performed undersubstantially isothermal conditions. Substantially isothermal conditionsinclude any conditions wherein the temperature is constrained within alimited range. In an exemplary embodiment, the temperature varies by nomore than 20° C., typically by no more than 10° C., 5° C. or 2° C.Isothermal amplification includes any amplification reaction wherein atleast two consecutive cycles of nucleic acid synthesis are performedunder substantially isothermal conditions, and include amplificationreactions wherein the temperature varies by no more than 20° C., 10° C.,5° C. or 2° C., over the duration of at least two consecutive cycles ofnucleic acid synthesis, although the temperature may vary by greaterthan 20° C. during the remainder of the amplification process, includingduring other cycles of nucleic acid synthesis. Optionally, in anisothermal reaction (including isothermal amplification), thetemperature is maintained at or around 50° C., 55° C., 60° C., 65° C.,or 70° C. for at least about 10, 15, 20, 30, 45, 60 or 120 minutes.Optionally, any temperature variation is not more than 20° C.,optionally within 10° C., for example within 5° C., or 2° C. during oneor more amplification cycles (e.g., e.g., 1, 5, 10, 20, or allamplification cycles performed). In some embodiments, isothermalamplification can include thermocycling, where temperature variance iswithin isothermal ranges. In an example, the temperature variation isconstrained between the denaturation step and another step such asannealing and/or extension. In an example, the difference between thedenaturation temperature and the annealing or extension temperature isnot more than 20° C., optionally within 10° C., for example within 5°C., or 2° C., for one or more cycles of amplification. The temperatureis optionally constrained for at least 5, 10, 15, 20, 30, 35 orsubstantially all cycles of amplification.

As used herein, the term “sequencing” and its variants compriseobtaining sequence information from a nucleic acid strand, typically bydetermining the identity of at least some nucleotides (including theirnucleobase components) within the nucleic acid molecule. While in someembodiments, “sequencing” a given region of a nucleic acid moleculeincludes identifying each and every nucleotide within the region that issequenced, “sequencing” can also include methods whereby the identity ofone or more nucleotides in is determined, while the identity of somenucleotides remains undetermined or incorrectly determined.

The terms “identity” and “identical” and their variants, as used herein,when used in reference to two or more nucleic acid sequences, refer tosimilarity in sequence of the two or more sequences (e.g., nucleotide orpolypeptide sequences). In the context of two or more homologoussequences, the percent identity or homology of the sequences orsubsequences thereof indicates the percentage of all monomeric units(e.g., nucleotides or amino acids) that are the same (i.e., about 70%identity, preferably 75%, 80%, 85%, 90%, 95% or 99% identity). Thepercent identity can be over a specified region, when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. Sequences are said to be“substantially identical” when there is at least 85% identity at theamino acid level or at the nucleotide level. Preferably, the identityexists over a region that is at least about 25, 50, or 100 residues inlength, or across the entire length of at least one compared sequence. Atypical algorithm for determining percent sequence identity and sequencesimilarity are the BLAST and BLAST 2.0 algorithms, which are describedin Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977). Other methodsinclude the algorithms of Smith & Waterman, Adv. Appl. Math. 2:482(1981), and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), etc.Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules or their complements hybridize toeach other under stringent hybridization conditions.

The term “complementary” and its variants, as used herein in referenceto two or more polynucleotides, refer to polynucleotides including anynucleic acid sequences (e.g., portions of target nucleic acid moleculesand primers) that can undergo cumulative base pairing at two or moreindividual corresponding positions in antiparallel orientation, as in ahybridized duplex. Optionally there can be “complete” or “totalcomplementarity between a first and second nucleic acid sequence whereeach nucleotide in the first nucleic acid sequence can undergo astabilizing base pairing interaction with a nucleotide in thecorresponding antiparallel position on the second nucleic acid sequence(however, the term “complementary” by itself can include nucleic acidsequences that are not completely complementary over their entirelength); “Partial” complementarity describes nucleic acid sequences inwhich at least 20%, but less than 100%, of the residues of one nucleicacid sequence are complementary to residues in the other nucleic acidsequence. In some embodiments, at least 50%, but less than 100%, of theresidues of one nucleic acid sequence are complementary to residues inthe other nucleic acid sequence. In some embodiments, at least 70%, 80%,90% or 95%, but less than 100%, of the residues of one nucleic acidsequence are complementary to residues in the other nucleic acidsequence. Sequences are said to be “substantially complementary” when atleast 85% of the residues of one nucleic acid sequence are complementaryto residues in the other nucleic acid sequence. “Noncomplementary”describes nucleic acid sequences in which less than 20% of the residuesof one nucleic acid sequence are complementary to residues in the othernucleic acid sequence. A “mismatch” is present at any position in thetwo opposed nucleotides are not complementary. Complementary nucleotidesinclude nucleotides that are efficiently incorporated by DNA polymerasesopposite each other during DNA replication under physiologicalconditions. In a typical embodiment, complementary nucleotides can formbase pairs with each other, such as the A-T/U and G-C base pairs formedthrough specific Watson-Crick type hydrogen bonding between thenucleobases of nucleotides and/or polynucleotides at positionsantiparallel to each other. The complementarity of other artificial basepairs can be based on other types of hydrogen bonding and/orhydrophobicity of bases and/or shape complementarity between bases.

The term “double stranded” and its variants, as used herein in referenceto any polynucleotide or nucleic acid molecule, refer to anypolynucleotide or nucleic acid molecule having one or more strands andincluding a region containing nucleotide residues that are base pairedwith nucleotide residues, for example as in a nucleic acid duplex.Optionally the double stranded polynucleotide (or nucleic acid molecule)can be “completely” or “totally” double stranded, such that eachnucleotide residue in the polynucleotide (or nucleic acid molecule) isbase paired with another corresponding nucleotide residue. In someembodiments, the double stranded polynucleotide includes one or moresingle stranded regions containing nucleotide residues that are not basepaired with any other nucleotide residue. In some embodiments, at least51%, 75%, 85%, 95%, 97% or 99%, of the nucleotide residues in the doublestranded polynucleotide (or nucleic acid molecule) are base paired withother nucleotide residues. In some embodiments, a double strandedpolynucleotide (or nucleic acid molecule) includes two strands that arenot covalently linked to each other; alternatively, the double strandedpolynucleotide (or nucleic acid molecule) includes a single strand thatis base paired with itself over at least some portion of its length asin, for example, a hairpin oligonucleotide. A polynucleotide is said tobe “substantially double stranded” when at least 85% of the nucleotideresidues of the polynucleotide are base paired with correspondingnucleotide residues. Two nucleic acid sequences are said to be “doublestranded” when the residues of one nucleic acid sequence are base pairedwith corresponding residues in the other nucleic acid sequence. In someembodiments, base pairing can occur according to some conventionalpairing paradigm, such as the A-T/U and G-C base pairs formed throughspecific Watson-Crick type hydrogen bonding between the nucleobases ofnucleotides and/or polynucleotides positions antiparallel to each other;in other embodiments, base pairing can occur through any other paradigmwhereby base pairing proceeds according to established and predictablerules.

As used herein, the term “single stranded” and its variants, when usedin reference to any polynucleotide or nucleic acid molecule, refer toany polynucleotide or nucleic acid molecule including a regioncontaining nucleotide residues that are not base paired with anynucleotide residues. Optionally the single stranded polynucleotide (ornucleic acid molecule) can be “completely” or “totally” single stranded,such that each nucleotide residue in the polynucleotide (or nucleic acidmolecule) is not base paired with any other nucleotide residue. In someembodiments, the single stranded polynucleotide includes one or moredouble stranded regions containing nucleotide residues that are basepaired with nucleotide residue. In some embodiments, at least 51%, 75%,85%, 95%, 97% or 99%, of the nucleotide residues in the single strandedpolynucleotide (or nucleic acid molecule) are not base paired with othernucleotide residues. A polynucleotide is said to be “substantiallysingle stranded” when at least 85% of the nucleotide residues of thepolynucleotide are not base paired with nucleotide residues.

As used herein, the term “denature” and its variants, when used inreference to any double stranded polynucleotide molecule, or doublestranded polynucleotide sequence, includes any process whereby the basepairing between nucleotides within opposing strands of the doublestranded molecule, or double stranded sequence, is disrupted. Typically,denaturation includes rendering at least some portion or region of twostrands of the double stranded polynucleotide molecule or sequencesingle stranded. In some embodiments, denaturation includes separationof at least some portion or region of two strands of the double strandedpolynucleotide molecule or sequence from each other. Typically, thedenatured region or portion is then capable of hybridizing to anotherpolynucleotide molecule or sequence. Optionally there can be “complete”or “total” denaturation of a double stranded polynucleotide molecule orsequence. Complete denaturation conditions are for example conditionsthat would result in complete separation of a significant fraction(e.g., more than 10%, 20%, 30%, 40% or 50%) of a large plurality ofstrands from their extended and/or full-length complements. Typically,complete or total denaturation disrupts all of the base pairing betweenthe nucleotides of the two strands with each other. Similarly a nucleicacid sample is optionally considered fully denatured when more than 80%or 90% of individual molecules of the sample lack anydouble-strandedness (or lack any hybridization to a complementarystrand).

Alternatively, the double stranded polynucleotide molecule or sequencecan be partially or incompletely denatured. A given nucleic acidmolecule can be considered partially-denatured when a portion of atleast one strand of the nucleic acid remains hybridized to acomplementary strand, while another portion is in an unhybridized state(even if it is in the presence of a complementary sequence). Theunhybridized portion is optionally at least 5, 7, 8, 10, 12, 15, 17, 20,or 50 nucleotides long. The hybridized portion is optionally at least 5,7, 8, 10, 12, 15, 17, 20, or 50 nucleotides long. Partial denaturationincludes situations where some but not all, of the nucleotides of onestrand or sequence, are based paired with some nucleotides of the otherstrand or sequence within a double stranded polynucleotide. In someembodiments, at least 20% but less than 100%, of the nucleotide residuesof one strand of the partially denatured polynucleotide (or sequence)are not base paired to nucleotide residues within the opposing strand.Under exemplary conditions at least 50% of nucleotide residues withinthe double stranded polynucleotide molecule (or double strandedpolynucleotide sequence) are in single stranded (or unhybridized) from,but less than 20% or 10% of the residues are double stranded.

Optionally, a nucleic acid sample can be considered to be partiallydenatured when a substantial fraction of individual nucleic acidmolecules of the sample (e.g., above 20%, 30%, 50%, or 70%) are in apartially denatured state. Optionally less than a substantial amount ofindividual nucleic acid molecules in the sample are fully denatured,e.g., not more than 5%, 10%, 20%, 30% or 50% of the nucleic acidmolecules in the sample. Under exemplary conditions at least 50% of thenucleic acid molecules of the sample are partly denatured, but less than20% or 10% are fully denatured. In other situations, at least 30% of thenucleic acid molecules of the sample are partly denatured, but less than10% or 5% are fully denatured. Similarly, a nucleic acid sample can beconsidered to be non-denatured when a minority of individual nucleicacid molecules in the sample are partially or completely denatured.

In an embodiment, partially denaturing conditions are achieved bymaintaining the duplexes as a suitable temperature range. For example,the nucleic acid is maintained at temperature sufficiently elevated toachieve some heat-denaturation (e.g., above 45° C., 50° C., 55° C., 60°C., 65° C., or 70° C.) but not high enough to achieve completeheat-denaturation (e.g., below 95° C. or 90° C. or 85° C. or 80° C. or75° C.). In an embodiment the nucleic acid is partially denatured usingsubstantially isothermal conditions.

Partial denaturation can also be achieved by other means, e.g., chemicalmeans using chemical denaturants such as urea or formamide, withconcentrations suitably adjusted, or using high or low pH (e.g., pHbetween 4-6 or 8-9). In an embodiment, partial denaturation andamplification is achieved using recombinase-polymerase amplification(RPA). Exemplary RPA methods are described herein.

In some embodiments, complete or partial denaturation is accomplished bytreating the double stranded polynucleotide sequence to be denaturedusing appropriate denaturing agents. For example, the double strandedpolynucleotide can be subjected to heat-denaturation (also referred tointerchangeably as thermal denaturation) by raising the temperature to apoint where the desired level of denaturation is accomplished. In someembodiments, complete thermal denaturation of a double strandedpolynucleotide, the temperature can be adjusted to achieve completeseparation of the two strands of the polynucleotide, such that at least90% of the strands are in single stranded form across their entirelength. In some embodiments, complete thermal denaturation of apolynucleotide molecule (or polynucleotide sequence) is accomplished byexposing the polynucleotide molecule (or sequence) to a temperature thatis at least 5° C., 10° C., 15° C., 20° C., 25, 30° C., 50° C., or 100°C., above the calculated or predict melting temperature (Tm) of thepolynucleotide molecule or sequence.

Alternatively, chemical denaturation can be accomplished by contactingthe double stranded polynucleotide to be denatured with appropriatechemical denaturants, such as strong alkalis, strong acids, chaotropicagents, and the like and can include, for example, NaOH, urea, orguanidine-containing compounds. In some embodiments, partial or completedenaturation is achieved by exposure to chemical denaturants such asurea or formamide, with concentrations suitably adjusted, or using highor low pH (e.g., pH between 4-6 or 8-9). In an embodiment, partialdenaturation and amplification is achieved using recombinase-polymeraseamplification (RPA). Exemplary RPA methods are described herein.

The terms “melting temperature”, “Tm” or “T_(m)” and their variants,when used in reference to a given polynucleotide (or a given targetsequence within a polynucleotide) typically refers to a temperature atwhich 50% of the given polynucleotide (or given target sequence) existsin double-stranded form and 50% is single stranded, under a defined setof conditions. In some embodiments, the defined set of conditions caninclude a defined parameter indicating ionic strength and/or pH in anaqueous reaction condition. A defined condition can be modulated byaltering the concentration of salts (e.g., sodium), temperature, pH,buffers, and/or formamide. Typically, the calculated thermal meltingtemperature can be at about 5-30° C. below the T_(m), or about 5-25° C.below the T_(m), or about 5-20° C. below the T_(m), or about 5-15° C.below the T_(m), or about 5-10° C. below the T_(m). Methods forcalculating a T_(m) are well known and can be found in Sambrook (1989 in“Molecular Cloning: A Laboratory Manual”, 2^(nd) edition, volumes 1-3;Wetmur 1966, J. Mol. Biol., 31:349-370; Wetmur 1991 Critical Reviews inBiochemistry and Molecular Biology, 26:227-259). Other sources forcalculating a T_(m) for hybridizing or denaturing nucleic acids includeOligoAnalyze (from Integrated DNA Technologies) and Primer3 (distributedby the Whitehead Institute for Biomedical Research). In someembodiments, the term “melting temperature”, “Tm” and “T_(m)” and theirvariants includes both the actual Tm of the given polynucleotide (ortarget sequence), as measured empirically using defined conditions, orthe predicted or calculated Tm. In some embodiments, the Tm of atemplate can be predicted or calculated without using the sequence ofthe template, by assuming that the template includes a certainproportion of the four common nucleotides (A, C, G and T) and has acertain length (or, in the case of a population of templates, an averagelength). For example, it can be assumed that a population of templates,that migrates as a smear on a gel, includes 25% each of A, C. G or T,and has an average length of 200, 300, 400 base pairs.

The term “label” and its variants, as used herein with respect to achemical moiety, includes any composition comprising an optically ornon-optically detectable moiety, where the detectable moiety has beenartificially added, linked or attached via chemical manipulation tosecond moiety that is unlabeled. Typically, addition of the label isperformed by a user (or by an upstream provider) with the purpose ofenhancing detectability of the second moiety. Optically or non-opticallydetectable components of a composition that already exist in thenaturally occurring form of the composition (for example, hydrogen ionsand amino acids present in a typical DNA molecule, an RNA molecule, or anucleotide within a natural cell) are not labels for purposes of thisdisclosure. Some typical labels include fluorescent moieties and dyes.

A nucleic acid can be considered immobilized if it is attached to asupport in a manner that is substantially stable, at least duringconditions of choice (e.g., during the amplification reaction). Theattachment can be by any mechanism, including but not limited tonon-covalent bonding, ionic interactions, covalent linkage. If a firstnucleic acid is hybridized to a second nucleic acid immobilized on asupport, then the first nucleic acid can also be considered to beimmobilized to the support during amplification, if amplificationconditions are such that substantial amounts of the first and secondnucleic acids are associated or connected with each other at any or alltimes during amplification. For example the first and second nucleicacids can be associated together by hybridization involving Watson-Crickbase pairing or hydrogen bonding. In an example, the amplificationconditions of choice allow at least 50%, 80%, 90%, 95% or 99% of thefirst nucleic acid to remain hybridized with the second nucleic acid, orvice versa. A nucleic acid can be considered unimmobilized ornon-immobilized if it is not directly or indirectly attached to orassociated with a support.

A medium can be considered flowable under conditions of choice if themedium is under those conditions at least temporarily a fluid mediumthat does not substantially or completely restrain or impede transfer ormovement of an unimmobilized molecule. The unimmobilized molecule is notitself immobilized to a solid support or surface or associated withanother immobilized molecule. In an embodiment, the unimmobilizedmolecule is a solute (e.g., a nucleic acid) through the flowable medium.Exemplary transfer or movement in the medium can be by means ofdiffusion, convection, turbulence, agitation, Brownian motion,advection, current flows, or other molecular movements within theliquid) from any first point in the continuous phase to any other pointin fluid communication or in the same continuous phase. For example, ina flowable medium a significant amount of an unimmobilized nucleic acidis transferred from one immobilization site to another immobilizationsite that is within the same continuous phase of the flowable medium, orin fluid communication with the first immobilization site. Optionally,the rate of transfer or movement of the nucleic acid in the medium iscomparable to the rate of transfer or movement of the nucleic acid inwater. In some instances, the conditions of choice are conditions thatthe medium is subjected to during amplification. The conditions ofchoice may or may not allow the flowable medium to remain substantiallymotionless. The conditions may or may not subject the flowable medium toactive mixing, agitation or shaking. The medium is optionally flowableat least temporarily during amplification. For example the medium isflowable under at least one preamplification and/or amplificationcondition of choice. Optionally, a flowable medium does notsubstantially prevent intermingling of different unimmobilized nucleicacids or transfer of an unimmobilized nucleic acid between differentzones of a continuous phase of the flowable medium. The movement ortransfer of nucleic acids for example can be caused by means ofdiffusion or convection. A medium is optionally considered nonflowableif unimmobilized nucleic acids upon amplification fail to spread or movebetween different immobilization sites or over the entire continuousphase. Generally, a flowable medium does not substantially confineunimmobilized nucleic acids (e.g., the templates or amplicons) withinlimited zones of the reaction volume or at fixed locations during theperiod of amplification. Optionally, a flowable medium can be renderednon-flowable by various means or by varying its conditions. Optionally,a medium is flowable if it is liquid or is not semisolid. A medium canbe considered flowable if its fluidity is comparable to pure water. Inother embodiments, a medium can be considered flowable if it a fluidthat is substantially free of polymers, or if its viscosity coefficientis similar to that of pure water.

As will be appreciated by one of ordinary skill in the art, referencesto templates, initializing oligonucleotides, extension probes, primers,etc., can refer to populations or pools of nucleic acid molecules thatare substantially identical within a relevant portion, rather thansingle molecules. For example, a “template” can refer to a plurality ofsubstantially identical template molecules; a “probe” can refer to aplurality of substantially identical probe molecules, etc. In the caseof probes that are degenerate at one or more positions, it will beappreciated that the sequence of the probe molecules that comprise aparticular probe will differ at the degenerate positions, i.e., thesequences of the probe molecules that constitute a particular probe maybe substantially identical only at the nondegenerate position(s). Theseterms within this application are intended to provide support for eithera population or a molecule. Where it is intended to refer to a singlenucleic acid molecule (i.e., one molecule), the terms “templatemolecule”, “probe molecule”, “primer molecule”, etc., may be usedinstead. In certain instances the plural nature of a population ofsubstantially identical nucleic acid molecules will be explicitlyindicated.

“Template”, “oligonucleotide”, “probe”, “primer”, “template”, “nucleicacid” and the like are intended to be interchangeable terms herein.These terms refer to polynucleotides, not necessarily limited to anylength or function. The same nucleic acid can be regarded as a“template”, “probe” or “primer” depending on the context, and can switchbetween these roles with time. A “polynucleotide,” also called a“nucleic acid,” is a linear polymer of two or more nucleotides joined bycovalent internucleosidic linkages, or variant or functional fragmentsthereof. In naturally occurring examples of these, the internucleosidelinkage is typically a phosphodiester bond. However, other examplesoptionally comprise other internucleoside linkages, such asphosphorothiolate linkages and may or may not comprise a phosphategroup. Polynucleotides include double- and single-stranded DNA, as wellas double- and single-stranded RNA, DNA:RNA hybrids, peptide-nucleicacids (PNAs) and hybrids between PNAs and DNA or RNA, and also includeknown types of modifications. Polynucleotides can optionally be attachedto one or more non-nucleotide moieties such as labels and other smallmolecules, large molecules such proteins, lipids, sugars, and solid orsemi-solid supports, for example through either the 5′ or 3′ end. Labelsinclude any moiety that is detectable using a detection method ofchoice, and thus renders the attached nucleotide or polynucleotidesimilarly detectable using a detection method of choice. Optionally, thelabel emits electromagnetic radiation that is optically detectable orvisible. In some cases, the nucleotide or polynucleotide is not attachedto a label, and the presence of the nucleotide or polynucleotide isdirectly detected. A “nucleotide” refers to a nucleotide, nucleoside oranalog thereof. Optionally, the nucleotide is an N- or C-glycoside of apurine or pyrimidine base. (e.g., deoxyribonucleoside containing2-deoxy-D-ribose or ribonucleoside containing D-ribose). Examples ofother analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides. Referring to a nucleic acid by any one ofthese terms should not be taken as implying that the nucleic acid hasany particular activity, function or properties. For example, the word“template” does not indicate that the “template” is being copied by apolymerase or that the template is not capable of acting as a “primer”or a “probe”.

It will be appreciated that in certain instances nucleic acid reagentsinvolved in amplification such as a template, probe, primer, etc., maybe a portion of a larger nucleic acid molecule that also containsanother portion that does not serve the same function. Optionally, thisother portion does not serve any template, probe, or primer function. Insome instances, a nucleic acid that substantially hybridizes to anoptionally-immobilized primer (e.g., on an immobilization site) isconsidered to be the “template”. Any one or more nucleic acid reagentsthat are involved in template walking (template, immobilized strands,immobilized or unimmobilized primer, etc.) may be generated before orduring amplification from other nucleic acids. The nucleic acid reagentis optionally generated from (and need not be identical to) an inputnucleic acid by making one or more modifications to the nucleic acidthat was initially introduced into the template walking medium. An inputnucleic acid can for example be subjected to restriction digestion,ligation, one or more amplification cycles, denaturation, mutation, etc,to generate a nucleic acid that serves as the template, primer, etc,during amplification or further amplification. For example, adouble-stranded input nucleic acid can be denatured to generate a firstsingle-stranded nucleic acid which optionally is used to generate asecond complementary strand. If so desired, the first single-strandednucleic acid can be considered the “template” for our purposes herein.Alternatively, the second complementary strand generated from the firstsingle-stranded nucleic acid can be considered the “template” for ourpurposes herein. In another example, a template is derived from an inputnucleic acid and is not necessarily identical to the input nucleic acid.For example, the template can comprise additional sequence not presentan input nucleic acid. In an embodiment the template can be an amplicongenerated from an input nucleic acid using one or more primers with a 5′overhang that is not complementary to the input nucleic acid.

The term “hybridize” and its variants, as used herein in reference totwo or more polynucleotides, refer to any process whereby any one ormore nucleic acid sequences (each sequence comprising a stretch ofcontiguous nucleotide residues) within said polynucleotides undergo basepairing at two or more individual corresponding positions, for exampleas in a hybridized nucleic acid duplex. Optionally there can be“complete” or “total” hybridization between a first and second nucleicacid sequence, where each nucleotide residue in the first nucleic acidsequence can undergo a base pairing interaction with a correspondingnucleotide in the antiparallel position on the second nucleic acidsequence. In some embodiments, hybridization can include base pairingbetween two or more nucleic acid sequences that are not completelycomplementary, or are not base paired, over their entire length. Forexample, “partial” hybridization occurs when two nucleic acid sequencesundergo base pairing, where at least 20% but less than 100%, of theresidues of one nucleic acid sequence are base paired to residues in theother nucleic acid sequence. In some embodiments, hybridization includesbase pairing between two nucleic acid sequences, where at least 50%, butless than 100%, of the residues of one nucleic acid sequence are basepaired with corresponding residues in the other nucleic acid sequence.In some embodiments, at least 70%, 80%, 90% or 95%, but less than 100%,of the residues of one nucleic acid sequence are base paired withcorresponding residues in the other nucleic acid sequence. Two nucleicacid sequences are said to be “substantially hybridized” when at least85% of the residues of one nucleic acid sequence are base paired withcorresponding residues in the other nucleic acid sequence. In situationswhere one nucleic acid molecule is substantially longer than the other(or where the two nucleic acid molecule include both substantiallycomplementary and substantially non-complementary regions), the twonucleic acid molecules can be described as “hybridized” even whenportions of either or both nucleic acid molecule can remainunhybridized. “Unhybridized” describes nucleic acid sequences in whichless than 20% of the residues of one nucleic acid sequence are basepaired to residues in the other nucleic acid sequence. In someembodiments, base pairing can occur according to some conventionalpairing paradigm, such as the A-T/U and G-C base pairs formed throughspecific Watson-Crick type hydrogen bonding between the nucleobases ofnucleotides and/or polynucleotides positions antiparallel to each other;in other embodiments, base pairing can occur through any other paradigmwhereby base pairing proceeds according to established and predictablerules.

Hybridization of two or more polynucleotides can occur whenever said twoor more polynucleotides come into contact under suitable hybridizingconditions. Hybridizing conditions include any conditions that aresuitable for nucleic acid hybridization; methods of performinghybridization and suitable conditions for hybridization are well knownin the art. The stringency of hybridization can be influenced by variousparameters, including degree of identity and/or complementarity betweenthe polynucleotides (or any target sequences within the polynucleotides)to be hybridized; melting point of the polynucleotides and/or targetsequences to be hybridized, referred to as “T_(m)”; parameters such assalts, buffers, pH, temperature, GC % content of the polynucleotide andprimers, and/or time. Typically, hybridization is favored in lowertemperatures and/or increased salt concentrations, as well as reducedconcentrations of organic solvents. High-stringency hybridizationconditions will typically require a higher degree of complementarybetween two target sequences for hybridization to occur, whereaslow-stringency hybridization conditions will favor hybridization evenwhen the two polynucleotides to be hybridized exhibit lower levels ofcomplementarity. The hybridization conditions can be applied during ahybridization step, or an optional and successive wash step, or both thehybridization and optional wash steps.

Examples of high-stringency hybridization conditions include any one ormore of the following: salt concentrations (e.g., NaCl) of from about0.0165 to about 0.0330; temperatures of from about 5° C. to about 10° C.below the melting point (T_(m)) of the target sequences (orpolynucleotides) to be hybridized; and/or formamide concentrations ofabout 50% or higher. Typically, high-stringency hybridization conditionspermit binding between sequences having high homology, e.g., ≥95%identity or complementarity. In one exemplary embodiment ofhigh-stringency hybridization conditions, hybridization is performed atabout 42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4),5×SSC, 5×Denhardt's solution, 50 μg/mL denatured, sonicated salmon spermDNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL double strandedpolynucleotide (or double stranded target sequence), while the washesare performed at about 65° C. with a wash solution containing 0.2×SSCand 0.1% sodium dodecyl sulfate.

Examples of medium-stringency hybridization conditions can include anyone or more of the following: salt concentrations (e.g., NaCl) of fromabout 0.165 to about 0.330; temperatures of from about 20° C. to about29° C. below the melting point (T_(m)) of the target sequences to behybridized; and/or formamide concentrations of about 35% or lower.Typically, such medium-stringency conditions permit binding betweensequences having high or moderate homology, e.g., ≥80% identity orcomplementarity. In one exemplary embodiment of medium stringencyhybridization conditions, hybridization is performed at about 42° C. ina hybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC, 5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50%formamide, 10% Dextran sulfate, and 1-15 ng/mL double strandedpolynucleotide (or double stranded target sequence), while the washesare performed at about 50° C. with a wash solution containing 2×SSC and0.1% sodium dodecyl sulfate.

Examples of low-stringency hybridization conditions include any one ormore of the following: salt concentrations (e.g., NaCl) of from about0.330 to about 0.825; temperatures of from about 40° C. to about 48° C.below the melting point (T_(m)) of the target sequences to behybridized; and/or formamide concentrations of about 25% or lower.Typically, such low-stringency conditions permit binding betweensequences having low homology, e.g., ≥50% identity or complementarity.

Some exemplary conditions suitable for hybridization include incubationof the polynucleotides to be hybridized in solutions having sodiumsalts, such as NaCl, sodium citrate and/or sodium phosphate. In someembodiments, hybridization or wash solutions can include about 10-75%formamide and/or about 0.01-0.7% sodium dodecyl sulfate (SDS). In someembodiments, a hybridization solution can be a stringent hybridizationsolution which can include any combination of 50% formamide, 5×SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, 0.1% SDS, and/or 10%dextran sulfate. In some embodiments, the hybridization or washingsolution can include BSA (bovine serum albumin). In some embodiments,hybridization or washing can be conducted at a temperature range ofabout 20-25° C., or about 25-30° C., or about 30-35° C., or about 35-40°C., or about 40-45° C., or about 45-50° C., or about 50-55° C., orhigher.

In some embodiments, hybridization or washing can be conducted for atime range of about 1-10 minutes, or about 10-20 minutes, or about 20-30minutes, or about 30-40 minutes, or about 40-50 minutes, or about 50-60minutes, or longer.

In some embodiments, hybridization or wash conditions can be conductedat a pH range of about 5-10, or about pH 6-9, or about pH 6.5-8, orabout pH 6.5-7.

In some embodiments, the term “monoclonal” and its variants is used todescribe a population of polynucleotides where a substantial portion ofthe members of the population (e.g., at least about 50%, typically atleast 75%, 80%, 85%, 90%, 95%, or 99%) share at least 80% identity atthe nucleotide sequence level. Typically, at least about 90% of thepopulation, typically at least about 95%, more typically at least about99%, 99.5% or 99.9%) are generated via amplification ortemplate-dependent replication of a particular polynucleotide sequence,which is present amongst a substantial portion of members of themonoclonal polynucleotide population. All members of a monoclonalpopulation need not be completely identical or complementary to eachother. For example, different portions of a polynucleotide template canbecome amplified or replicated to produce the members of the resultingmonoclonal population; similarly, a certain number of “errors” and/orincomplete extensions may occur during amplification of the originaltemplate, thereby generating a monoclonal population whose individualmembers can exhibit sequence variability amongst themselves. In someembodiments, at least 50% of the members of the monoclonal populationare at least 80% identical to a reference nucleic acid sequence (i.e., anucleic acid of defined sequence used as a basis for a sequencecomparison). In some embodiments, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 99%, or more of the members ofa population include a sequence that is at least 80%, 85%, 90%, 95%, 97%or 99% identical (or complementary) to the reference nucleic acidsequence. In some embodiments, a low or insubstantial level of mixing ofnon-homologous polynucleotides may occur during nucleic acidamplification reactions described herein, and thus a substantiallymonoclonal population may contain a minority of diverse polynucleotides(e.g., less than 30%, less than 20%, less than 10%, less than 5%, lessthan 1%, less than 0.5%, less than 0.1%, or less than 0.001%, of diversepolynucleotides). As used herein, the phrase “substantially monoclonal”and its variants, when used in reference to one or more polynucleotidepopulations, refer to one or more polynucleotide populations comprisedof polynucleotides that are at least 80% identical to the originalsingle template used as a basis for clonal amplification to produce thesubstantially monoclonal population.

In some embodiments, at least 80% of the members of the amplicon,typically at least 90%, more typically at least 95%, even more typicallyat least 99%, of the members of the amplicon will share greater than 90%identity, typically greater than 95% identity, more typically greaterthan 97%, and even more typically greater than 99% identity with thepolynucleotide template. Alternatively, the members of the amplicon canbe greater than 90% complementary, typically greater than 95%complementary, more typically greater than 97% complementary, even moretypically greater than 99% complementary, to the original template. Insome embodiments, members of a substantially monoclonal nucleic acidpopulation can hybridize to each other under high stringencyhybridization conditions.

In some embodiments, an amplicon is referred to as “monoclonal” or“substantially monoclonal” if it includes sufficiently few polyclonalcontaminants to produce a detectable signal in any method of nucleicacid analysis that is influenced by the sequence of the template. Forexample, a “monoclonal” population of polynucleotides can include anypopulation that produces a signal (e.g., a sequencing signal, anucleotide incorporation signal and the like) that can be detected usinga particular sequencing system. Optionally, the signal can subsequentlybe analyzed to correctly determine the sequence and/or base identity ofany one or more nucleotides present within any polynucleotide of thepopulation. Examples of suitable sequencing systems for detection and/oranalysis of such signals include the Ion Torrent sequencing systems,such as the Ion Torrent PGM™ sequence systems, including the 314, 316and 318 systems, and the Ion Torrent Proton™ sequencing systems,including Proton I, Proton II and Proton III (Life Technologies,Carlsbad, Calif.). In some embodiments, the monoclonal amplicon permitsthe accurate sequencing of at least 5 contiguous nucleotide residues onan Ion Torrent sequencing system.

As used herein, the term “clonal amplification” and its variants referto any process whereby a substantially monoclonal polynucleotidepopulation is produced via amplification of a polynucleotide template.In some embodiments of clonal amplification, two or more polynucleotidetemplates are amplified to produce at least two substantially monoclonalpolynucleotide populations.

As used herein, the term “adaptor” includes polynucleotides oroligonucleotides comprising DNA, RNA, chimeric RNA/DNA molecules, oranalogs thereof and typically refers to an added or extraneous sequencethat is linked or attached to the target polynucleotide of interest(e.g., the template) during the course of manipulation. Ligation of theadapter to the template can optionally occur prior to or after templateamplification. In some embodiments, the adapter can include a primerbinding sequence that is substantially identical, or substantiallycomplementary, to a sequence within a corresponding primer. In someembodiments, a first adapter including a first primer binding site isligated to one end of a linear double stranded template, while a secondadapter including a second primer binding site is ligated to the otherend.

As used herein, the term “binding partners” includes two molecules, orportions thereof, which have a specific binding affinity for one anotherand typically will bind to each other in preference to binding to othermolecules. Typically but not necessarily some or all of the structure ofone member of a specific binding pair is complementary to some or all ofthe structure possessed by the other member, with the two members beingable to bind together specifically by way of a bond between thecomplementary structures, optionally by virtue of multiple noncovalentattractions.

In some embodiments, molecules that function as binding partnersinclude: biotin (and its derivatives) and their binding partner avidinmoieties, streptavidin moieties (and their derivatives); His-tags whichbind with nickel, cobalt or copper; cysteine, histidine, or histidinepatch which bind Ni-NTA; maltose which binds with maltose bindingprotein (MBP); lectin-carbohydrate binding partners; calcium-calciumbinding protein (CBP); acetylcholine and receptor-acetylcholine; proteinA and binding partner anti-FLAG antibody; GST and binding partnerglutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNAglycosylase inhibitor) protein; antigen or epitope tags which bind toantibody or antibody fragments, particularly antigens such asdigoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and theirrespective antibodies; mouse immunoglobulin and goat anti-mouseimmunoglobulin; IgG bound and protein A; receptor-receptor agonist orreceptor antagonist; enzyme-enzyme cofactors; enzyme-enzyme inhibitors;and thyroxine-cortisol. Another binding partner for biotin can be abiotin-binding protein from chicken (Hytonen, et al., BMC StructuralBiology 7:8).

An avidin moiety can include an avidin protein, as well as anyderivatives, analogs and other non-native forms of avidin that can bindto biotin moieties. Other forms of avidin moieties include native andrecombinant avidin and streptavidin as well as derivatized molecules,e.g. nonglycosylated avidins, N-acyl avidins and truncatedstreptavidins. For example, avidin moiety includes deglycosylated formsof avidin, bacterial streptavidins produced by Streptomyces (e.g.,Streptomyces avidinii), truncated streptavidins, recombinant avidin andstreptavidin as well as to derivatives of native, deglycosylated andrecombinant avidin and of native, recombinant and truncatedstreptavidin, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyland N-succinyl avidin, and the commercial products ExtrAvidin™,Captavidin™, Neutravidin™ and Neutralite Avidin™.

EXAMPLES

Embodiments of the present teachings can be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1

A nucleic acid amplification reaction was conducted in single reactionvessel in a single continuous liquid phase in a total reaction volume of˜220 μL.

About 420 million beads were washed with water xl time (vortex/spin)then washed in buffer ×1 time (vortex/spin) in a 1.5 mL tube (tube 1).

The recombinase source was from a TwistAmp™ Basic kit (from TwistDx,Cambridge, Great Britain). Dehydrated pellets in the kit contain usvXrecombinase, usvY recombinase loading protein, gp32 protein, Bsu DNApolymerase, dNTPs, ATP, phosphocreatine and creatine kinase. Fourpellets from a TwistAmp™ Basic kit were rehydrated in 120 μL ofRehydration buffer supplied from the kit (tube 2). The recombinasesolution was vortexed and spun, then iced. Two heat blocks wereprepared, one set at about 68-70° C., and one at 40° C.

The supernatant was removed from the bead pellet (tube 1) leaving about˜20 μL of liquid at the bottom.

A reverse primer (2 μL of a 100 μM stock) was added to the bead tube(tube 1), then vortexed and spun. The reverse primer sequence:5′-ATCCCTGCGTGTCTCCGAC-3.

A biotinylated reverse primer (2 uL of a 10 μM stock) was added to thebead tube (tube 1), then vortexed and spun. The biotinylated reverseprimer sequence: 5′Bio-ATCCCTGCGTGTCTCCGAC-3′.

One μL of polynucleotide library (at various concentrations) was addedto the bead tube (tube 1), and vortexed/spun, and placed on ice. Thelibrary concentration varied depending on the desired DNA-to-bead ratioof 1:50, 1:75, 1:200.

The rehydrated recombinase mix (tube 2, reconstituted in 120 μLrehydration buffer) was added to the bead tube (tube 1), and vortexed,spun; and placed on ice.

65 μL of an exemplary sieving agent of the disclosure was added to thebead tube, vortexed, and spun, and put on ice.

11 μL of iced 280 mM Mg-acetate was added to the bead tube (in themiddle), then vortexed 3 seconds at a maximum setting and put back onice for 10 seconds, and incubated at 40° C. for 20 minutes on the heatblock.

The reaction was heat-killed at 68° C.-70° C. for 10 minutes in the heatblock.

The reaction tube was topped off with TE buffer, vortexed and spun at amaximum setting (˜20KG) for 3 minutes, the solution was removed from thebeads, leaving ˜100 μL. The wash step was repeated two times.

The beads were washed once with recovery solution.

The reaction tube was topped off with wash buffer, vortexed and spun ata maximum setting (˜20KG) for 3 minutes, the solution was removed fromthe beads, leaving ˜100 μL. The wash step was repeated two times.

After the last spin, bring the solution down to 100 μL (wash solution).

The beads were enriched by binding the biotinylated polynucleotides withparamagnetic beads conjugated with streptavidin (MyOne™ Bead fromDynabeads).

The enriched beads were loaded into an Ion Torrent ion-sensitive chipand a standard sequencing reaction was conducted. A significant portionof the enriched beads were determined to include a substantiallymonoclonal population of amplified polynucleotides, as evidenced by theobservation of detectable sequencing signals on the Ion Torrent PGM™sequencer from such beads. The sequencing signals were analyzed todetermine a sequence present within the amplicon of each such bead.

Example 2

About 240 million beads (attached with forward primers) were washed oncein annealing buffer (from Ion Sequencing kit, e.g., PN 4482006) in a 2mL tube. The supernatant was removed (except ˜50 μL) and discarded. Thebeads were resuspended in 100 μL annealing buffer.

Barcoded DNA libraries having either 300 bp or 400 bp insert (about120-240 million copies) was pre-hybridized with the washed beads. Thelibrary included an insert sequence joined at one end to an adaptor thathybridizes to a forward primer and joined at the other end to an adaptorthat hybridizes to a reverse primer. The template/bead ratios testedincluded 1:1, 0.75:1, and 0.5:1. The final volume was adjusted to 200 μLwith annealing buffer. The tube was mixed by vortexing and spun. Thetube was incubated at 95-100° C. for 3 minutes, and at 37° C. for 5minutes. One mL of annealing buffer was added, the tube was vortexed andspun at more than 16,000×G for 3.5 minutes, and the supernatant wasdiscarded. One mL of 10 mM potassium-acetate was added, the tube wasvortexed and spun at more than 16,000×G for 3.5 minutes, and thesupernatant was discarded. The potassium-acetate wash was repeated once.The beads were resuspended in 480 μL of potassium acetate (tube 1).

The recombinase source was from a TwistAmp′ Basic kit (from TwistDx,Cambridge, Great Britain). See Example 1 above for a list of componentsin the dehydrated pellets from the Basic kit. In a 15 mL tube (tube 2),96 pellets from a TwistAmp′ Basic kit were rehydrated in 2.88 mL ofRehydration buffer supplied from the kit.

48 μL of 100 μM reverse primers (non-immobilized primers) were added tothe washed/pre-hybridized beads (tube 1). 48 μL of 10 μM biotinylatedreverse primers (non-immobilized primers) were added to thewashed/pre-hybridized beads, and the tube was vortexed (tube 1). Thecontents of tube 1 (containing the library, beads and reverse primers)was added to tube 2 (containing rehydrated pellets), and tube 2 wasvortexed for 5 seconds and placed on ice. 144 μL of T4 gp32 protein (15μg/μL) was added, and vortexed and returned to ice. 1.56 mL of anexemplary sieving agent of the disclosure was added, the tube wasvortexed and returned to ice. After the reaction remained on ice formore than 5 minutes, 264 μL of magnesium acetate was added, the tube wasvortexed three times for 3 seconds each. 50 μL samples were aliquotedinto an ice-chilled 96-well plate. The 96-well plate was incubated at40° C. for 25 minutes on a thermo-cycler (the temperature was sustainedat 40° C.).

To stop the reaction, 150 μL of 100 mM EDTA was added to each well. Allthe reactions were pooled and centrifuged at more than 16,000×G for 3.5minutes. The supernatant was discarded. 1 mL of Tris/1% SDS was added,and the tube was vortexed. The beads were washed twice in 1 mL OneTouchwash solution. The beads were resuspended in 100 μL.

Beads templated with copies of the library were enriched by binding withparamagnetic streptavidin beads (MyOne™ beads from Dynabeads). Theenriched beads were loaded into an Ion Torrent PGM ion-sensitive chip.

A standard sequencing reaction was conducted according to manufacturer'sinstructions in an Ion PGM™ Sequencing 400 Kit (User Guide PN4474246B).A significant portion of the enriched beads that were loaded onto thechip were determined to include substantially monoclonal populations ofamplified polynucleotides, as evidenced by the observation of detectablesequencing signals on the Ion Torrent PGM™ sequencer from these beads.The sequencing signals were analyzed by Torrent Suite Software todetermine the sequence present within the amplicon of these beads.

The sequencing data yielded 305 bp mean read length (FIG. 9 ), and thealigned quality measurements were 1.16 G (AQ17) and 1.07 G (AQ20).

Example 3

About 250 million beads (attached with forward primers) were washed oncein 1.5 mL of annealing buffer (from Ion Sequencing kit, e.g., PN4482006), vortexed, and spun at 15,000×G for 6 minutes. The supernatantwas discarded leaving about 50 μL in the tube.

A library having 140 bp insert (about 50 million copies) waspre-hybridized with the washed beads. The library included an insertsequence joined at one end to an adaptor that hybridizes to a forwardprimer and joined at the other end to an adaptor that hybridizes to areverse primer. The library (0.81 μL of a 62 M stock) and 0.1 mLAnnealing buffer was added to the washed beads and mixed by pipetting upand down. The bead/template ratio was about 5:1. The tube was incubatedat 92-95° C. for 7 minutes, and at 37° C. for 10 minutes. One mL ofannealing buffer was added, the tube was vortexed and spun at more than15,000×G for 6 minutes, and the supernatant was discarded. One mL of 10mM potassium-acetate was added, the tube was vortexed and spun at morethan 15,000×G for 6 minutes, and the supernatant was discarded. Thepotassium-acetate wash was repeated once and the tube placed on ice.About 60 of liquid remained in the tube (tube 1).

The recombinase source was from a TwistAmp™ Basic kit (from TwistDx,Cambridge, Great Britain). See Example 1 above for a list of componentsin the dehydrated pellets from the Basic kit. 8 pellets from a TwistAmp™Basic kit were rehydrated in about 240 of Rehydration buffer suppliedfrom the kit. The pellets and rehydration buffer were vortexed, spun andiced.

4 μL of 100 μM reverse primers (non-immobilized primers) and 1 μL of 10μM biotinylated reverse primers (non-immobilized primers) were added tothe washed/pre-hybridized beads (tube 1), and the tube was vortexed andspun (tube 1). The contents of tube 1 (containing the library, beads andreverse primers) was added to tube 2 (containing rehydrated pellets),and tube 2 was vortexed and placed on ice. 130 μL of an exemplarysieving agent of the disclosure was added, the tube was vortexed andreturned to ice. 24 μL of ice cold 280 mM magnesium acetate was added,the tube was vortexed and spun. The total reaction volume was about 332μL. The tube was incubated at 40° C. for 60 minutes.

1 ml of 100 mM EDTA was added to stop the reaction. The tube wasvortexed and spun at 15,000×G for 6 minutes. The supernatant wasdiscarded leaving about 20 μL in the tube. The EDTA stop reaction step,vortexing and spinning steps were repeated. 1 mL of Tris/1% SDS wasadded, and the tube was vortexed and spun at 15,000×G for 6 minutes. Thesupernatant was discarded leaving about 50 μL in the tube. The beadswere washed in 1 mL OneTouch wash solution by vortexing and spinning,leaving about 100 μL in the tube. All reactions were pooled and spun at15,000×G for 6 minutes, the supernatant was discarded, leaving about 100μL in the tube.

Beads templated with copies of the library were enriched by binding withparamagnetic streptavidin beads (MyOne™ beads from Dynabeads). Theenriched beads were loaded into an Ion Torrent Proton I™ ion-sensitivechip.

A standard sequencing reaction was conducted according to manufacturer'sinstructions in an Ion PI™ Sequencing 200 Kit (User Guide PNMAN0007491). A significant portion of the enriched beads that wereloaded onto the chip were determined to include substantially monoclonalpopulations of amplified polynucleotides, as evidenced by theobservation of detectable sequencing signals on the Ion Torrent Proton™sequencer from these beads. The sequencing signals were analyzed byTorrent Suite Software to determine the sequence present within theamplicon of these beads.

Two sequencing runs were performed. The sequencing data yielded 96 bpmean read length for the first run (FIG. 10 ) and 94 bp read length forthe second run (FIG. 11 ). The aligned quality measurements were 1.76 G(AQ17) and 1.43 G (AQ20) for the first run, and 1.48 G (AQ17) and 1.17 G(AQ20) for the second run.

Example 4

20 μL of beads (attached with forward primers) (103 million/μL) wasmixed with 440 μL of 10 mM potassium acetate and 3 μL of 1 M Tris (pH8). The beads were mixed by vortexing and spun down.

16 μL of a library having 200 bp insert (about 199 million copies) wasdenatured by mixing with 2 μL of NaOH, vortexed and spun, and allowed tosit for 1 minute. The reaction was neutralized by adding 440 μL of 10 mMpotassium acetate and 3 μL of 1M Tris pH 8. The library included aninsert sequence joined at one end to an adaptor that hybridizes to aforward primer and joined at the other end to an adaptor that hybridizesto a reverse primer.

The beads were added to the denatured library. The bead/template ratiowas about 10:1 (200 billion beads:200 million library). The tube wasvortexed, and allowed to sit at room temperature for 5 minutes (tube 1).

The recombinase source was from a TwistAmp™ Basic kit (from TwistDx,Cambridge, Great Britain). See Example 1 above for a list of componentsin the dehydrated pellets from the Basic kit. In a 15 mL tube (tube 2),96 pellets from a TwistAmp′ Basic kit were rehydrated in 3 mL ofRehydration buffer supplied from the kit (tube 2).

24 μL of 100 μM reverse primers (non-immobilized primers) and 2 μL of100 μM biotinylated reverse primers (non-immobilized primers) were addedto the washed/pre-hybridized beads, and the tube was vortexed (tube 1)and iced. 1.6 mL of an exemplary sieving agent of the disclosure wasadded to tube 2, the tube was vortexed 5 seconds, rotated/spin by handfor 10 seconds, vortexed for 5 seconds, and placed on ice. 260 μL of 280mM magnesium acetate was added to tube 2, the tube was vortexed 5seconds and rotated/spin by hand for 10 seconds (vortex and rotate/spinthree times), placed on ice. 50 μL samples were aliquoted into anice-chilled 96-well plate. The 96-well plate was incubated at 40° C. for60 minutes.

100 μL of 200 mM EDTA was added to each well to stop the reaction. Allthe reactions were pooled and centrifuged at maximum speed for 7minutes. The supernatant was discarded. The pellets were resuspended in1 mL Recovery buffer with 1% SDS, vortexed for 30 seconds, and spun atmaximum speed for 6 minutes. After every spin, the tubes were reduced byhalf by pooling the contents of two tubes. The pellets were resuspendedin 1 mL Recovery buffer with 1% SDS, vortexed for 30 seconds, and spunat 1550 rpm for 7 minutes.

Beads templated with copies of the library were enriched by binding withparamagnetic streptavidin beads (MyOne™ beads from Dynabeads). Duringthe enriching step, ES-wash buffer was replaced with Recovery bufferwith 0.1% SDS. The beads were finally resuspended in 1 mL water andreduced to 100 μL. The enriched beads were loaded into an Ion TorrentProton I™ ion-sensitive chip.

A standard sequencing reaction was conducted according to manufacturer'sinstructions in an Ion PI™ Sequencing 200 Kit (User Guide PNMAN0007491). A significant portion of the enriched beads that wereloaded onto the chip were determined to include substantially monoclonalpopulations of amplified polynucleotides, as evidenced by theobservation of detectable sequencing signals on the Ion Torrent Proton™sequencer from these beads. The sequencing signals were analyzed byTorrent Suite Software to determine the sequence present within theamplicon of these beads.

The sequencing data yielded 144 bp mean read length (FIG. 12 ), and thealigned quality measurements was 4 G (AQ17).

Example 5

375 million beads (attached with forward primers) were washed in dH₂O byvortexing and spinning. The supernatant was removed (except ˜50 μL). ADNA library (about 75 million molecules) was added to the washed beads.About 4 μL of reverse primer (non-biotinylated) and 0.4 μL ofbiotinylated reverse primer was added to the beads. About 0.8 μL of afusion forward primer was added to the beads. Forty μL of magnesiumacetate was added to the beads (final concentration 14 mM). dH₂O wasadded to the beads to a final total volume of 320 μL.

The recombinase source was from a TwistAmp′ Basic kit (from TwistDx,Cambridge, Great Britain). See Example 1 above for a list of componentsin the dehydrated pellets from the Basic kit. In a separate tube, 16pellets from a TwistAmp′ Basic kit were rehydrated in 488 μL ofRehydration buffer supplied from the kit. For 400 bp libraries, 0.25mg/ml of T4 gp32 protein (0.2 mg final concentration) was added, or for600 bp libraries, 0.5 mg/ml of T4 gp32 protein (0.4 mg finalconcentration) was added. The tube was vortexed to mix and spun.

The contents of the bead mixture was added to the recombinase tube,vortexed and spun. The bead/recombinase mixture was transferred to atube with chilled oil and assembled on a 10 micron Sterlitech filter onan Ion Torrent OneTouch™ apparatus. Emulsions were generated and brokenaccording to manufacture's instructions.

Beads templated with copies of the library were enriched (or theenriching step was omitted) by binding with paramagnetic streptavidinbeads (MyOne™ beads from Dynabeads). The enriched beads were loaded intoan Ion Torrent ion-sensitive chip and a standard sequencing reaction wasconducted. A significant portion of the enriched beads were determinedto include a substantially monoclonal population of amplifiedpolynucleotides, as evidenced by the observation of detectablesequencing signals on the Ion Torrent PGM™ sequencer from such beads.The sequencing signals were analyzed to determine a sequence presentwithin the amplicon of such beads.

Example 6

About 120 million beads (attached with forward primers) was washed oncewith annealing buffer. The supernatant was removed (except ˜50 μL). Thewashed beads were resuspended in 100 μL of annealing buffer. About 60million molecules of DNA library was added to the beads. The finalvolume was adjusted to 200 μL with annealing buffer. The beads andlibrary were mixed by vortexing and spinning. The bead/library mix washeated to 95-100° C. for 3 minutes, then incubated at 37° C. for 5minutes.

One mL of 10 mM potassium acetate was added to the bead/library mix,then vortexed and spun. The supernatant was discarded. The potassiumacetate wash was repeated once. The bead/DNA was resuspended in 120 μLpotassium acetate.

The recombinase source was from a TwistAmp™ Basic kit (from TwistDx,Cambridge, Great Britain). See Example 1 above for a list of componentsin the dehydrated pellets from the Basic kit. In a separate tube, 24pellets from a TwistAmp™ Basic kit were rehydrated in 720 μL ofRehydration buffer supplied from the kit. An additional 54 μL of dNTPmix (containing 10 mM each dNTP) was added to the TwistAmp′ mix.

In a third tube, 3 μL of streptavidin (500 μM) was mixed with 12biotinylated reverse primers (100 then transferred to the bead/librarytube. Recombinase mix was added to the bead/library tube, then vortexedto mix, and iced. 27 μL of T4 gp 32 protein (15/μg/μL) was added to thebead/library tube, then vortexed to mix. 390 μL of an exemplary sievingagent was added to the bead/library tube, the tube was inverted andvortexed to mix, and iced for at least 5 minutes. 80 μL of magnesiumacetate was added to the bead/library tube, and the tube was vortexed,spun, and iced for at least 10 seconds. The bead/library tube wasincubated at 40° C. for 40 minutes. The reaction was stopped by adding500 μL EDTA (250 mM). The tube was spun at greater than 18,000×G for 3minutes. The supernatant was discarded, the pellet was resuspended in 1mL TE with 1% SDS. The pellet resuspended by pipetting up and down, andwashed by adding 2 mL Ion Torrent OneTouch™ wash solution. The wash stepwas repeated once. The pellet was resuspended in 300 μL of melt-offsolution, and incubated with rocking for 5 minutes.

Beads templated with copies of the library were enriched (or theenriching step was omitted) by binding with paramagnetic streptavidinbeads (MyOne™ beads from Dynabeads). The enriched beads were loaded intoan Ion Torrent ion-sensitive chip and a standard sequencing reaction wasconducted. A significant portion of the enriched beads were determinedto include a substantially monoclonal population of amplifiedpolynucleotides, as evidenced by the observation of detectablesequencing signals on the Ion Torrent PGM™ sequencer from such beads.The sequencing signals were analyzed to determine a sequence presentwithin the amplicon of such beads.

Example 7

Nucleic acid amplification was conducted on an Ion sequencing chip.First a template walking amplification reaction was performed on thechip followed by a recombinase-mediated amplification reaction.

An Ion Torrent PGM™ sequencing chip was prepared to contain low T_(M)single-stranded primers attached at their 5′ ends to the floor of thewells. The immobilized primers contained a polyA(30) sequence.

The double-stranded DNA template included a single-stranded terminaloverhang sequence having a polyT(30) sequence.

Ion Torrent PGM™ sequencing chips were treated with a polymer to producea matrix at the bottom of the wells. Capture primers were attached tothe matrix.

The Ion Torrent PGM™ sequencing chip was pre-washed once with aTE-containing buffer, and vacuumed dry.

Forty microliters of a solution was mixed and loaded onto the chip. Thefinal concentration of the solution contained: 1λ Isothermal buffer fromNew England Biolabs, 1.6 mM MgSO₄, 3 mM dNTPs, 1 U/uL Bst polymerase(e.g., from New England Biolabs), 0.1 nM template, and nuclease-freewater to 40 uL volume. The chip was centrifuged for 5 minutes andincubated at 37° C. for 30 minutes. The chip was vacuumed dry.

Template walking amplification: Forty microliters of a template walkingsolution was loaded onto the chip. The final concentration of thetemplate walking solution contained: 1× Isothermal buffer from NewEngland Biolabs, 3.6 mM MgSO₄, 5 mM dNTPs, 2 uM soluble single-strandedprimer, 6 U/uL Bst polymerase (from New England Biolabs), andnuclease-free water to 40 uL volume. The chip was centrifuged, andincubated at 60° C. for 30 minutes. The chip was washed once with1×TE-containing buffer and vacuumed dry.

Recombinase-mediated amplification: The recombinase source for thisexample was from a TwistAmp™ Basic kit (from TwistDx, Cambridge, GreatBritain). See Example 1 above for a list of components in the dehydratedpellets from the Basic kit. Fifty microliters of an amplificationreaction mixture (containing recombinase) was loaded onto the chip. Theamplification reaction mixture contained: one pellet from a TwistAmp™Basic kit (from TwistDx, Cambridge, Great Britain), 30 uL rehydrationbuffer from the TwistAmp™ Basic kit, 2 uM of a soluble primer thathybridizes with one adaptor of the DNA template, and nuclease-free waterto 50 uL total volume. The chip was centrifuged for 2 minutes. Twomicroliters of magnesium acetate (280 mM stock) was added to the chip.The chip was centrifuged for 2 minutes, then incubated at 40° C. for 1hour.

The chip was washed sequentially with 0.5 M EDTA (pH 8), TE-containingbuffer, 1% SDS, and washed 2× with wash solution.

A majority of the wells were determined to include substantiallymonoclonal populations of amplified polynucleotides using a color-codedalignment map of the chip.

A standard sequencing reaction was conducted according to manufacturer'sinstructions in an Ion PI™ Sequencing 200 Kit (User Guide PNMAN0007491). The sequencing signals were analyzed by Torrent SuiteSoftware to determine the sequence present within the amplicon of thesebeads. The sequencing data yielded 151 bp mean read length.

Example 8

Nucleic acid amplification was performed directly on an Ion Torrent PGM™sequencing chip in the presence of recombinase, under isothermalconditions. A polymerized hydrogel was deposited in the wells of a PGM™chip.

The recombinase source for this example was from a TwistAmp™ Basic kit(from TwistDx, Cambridge, Great Britain). See Example 1 above for a listof components in the dehydrated pellets from the Basic kit.

A polynucleotide template was denatured using a heat denaturation methodor the recombinase method (described below), then proceeded to thenucleic amplification step.

(A) Heat denaturation method: a polynucleotide template library wasdiluted into a final volume of 60 μL in annealing buffer. The dilutiontargeted depositing about five copies of the template per well of an IonTorrent Proton™ sequencing chip (about 600-650 million wells). The chipwas washed once with annealing buffer, and set on a thermocycler set at40° C. An aliquot of 100 μL of 1:1 annealing buffer-to-water ratio wasmixed and pre-heated at 95° C. The template library was denatured bydeposition onto the chip and incubating at 95° C. for 2 minutes. Thebuffer/water mixture (pre-heated to 95° C.) was pipette into theflowcell. The chip was transferred to the 40° C. thermocycler andincubated for 5 minutes. The chip was transferred to the bench top(approximately 25° C.). The chip was washed with 100 μL of annealingbuffer. The chip was placed on a 4° C. thermocycler. The following areoptional steps: 1 pellet from a TwistAmp′ Basic kit was rehydrated in 20μL of water and 30 μL of Rehydration buffer supplied from the kit. Thepellet mix was vortexed vigorously to dissolve the pellet. 50 μL of thepellet mix was loaded onto the chip. The chip was incubated at roomtemperature for at least one minute to allow the recombinase to bind theprimers pre-loaded into the wells of the chip.

(B) Recombinase denaturation method: an Ion Torrent Proton™ sequencingchip (about 600-650 million wells) was washed with 150 μL of anannealing/water mix (1:1 ratio of annealing buffer and water). The chipwas set on a 40° C. thermocycler. 2 pellets from a TwistAmp™ Basic kitwas rehydrated in 60 μL of Rehydration buffer supplied from the kit. Apolynucleotide template library was diluted into a final volume of 50 μLin annealing buffer. The dilution targeted depositing about five copiesof the template per well of an Ion Torrent Proton™ sequencing chip. Thetotal volume of the diluted template was added to the rehydrated pelletmix. The volume was adjusted to 100 μL with water. The pellet/templatewas vortexed to mix and spun. The pellet/template mix was loaded ontothe Ion Torrent Proton™ sequencing chip (set on the 40° C. thermocycler)and incubated for 20 minutes. The chip was removed from the thermocyclerand set at room temperature.

Nucleic acid amplification was performed as follows: All reagents werekept on ice. One pellet from a TwistAmp′ Basic kit were rehydrated in 30μL of Rehydration buffer supplied from the kit, 16 μL of nuclease-freewater, and 1 μL of 100 μM reverse amplification primer. The pellet wasdissolved by vortexing and spinning. Immediately prior to loading ontothe chip, 3 μL of 280 μM of magnesium acetate was added to the pelletmix. The entire pellet mix was loaded onto the chip and incubated at 40°C. for 1 hour.

The amplification reaction was stopped by washing the chip with 0.1 MEDTA (pH 8). The chip was washed with a chip wash solution. The chip waswashed with 1% SDS. The chip was washed twice with TEX wash solution.

The chip was prepared for sequencing: The chip was washed with melt offsolution. The chip was washed three times with annealing buffer andplaced on a 40° C. thermocycler. In a separate tube the sequencingprimers were prepared: 80 μL of 50% annealing buffer was mixed with 50%sequencing primer, then preheated to 95° C. A 1:1 mixture of annealingbuffer and water was prepared and pre-heated to 95° C. The chip waswashed with pre-heated anneling/water buffer. The chip was loaded with80 μL of the pre-heated primer mix. The chip was incubated for 5 minutesat 40° C. The chip was washed once with the annealing/water buffer. In aseparate tube, 6 μL of sequencing polymerase was mixed with 57 μL ofannealing/water mixture, and loaded onto the chip.

A standard sequencing reaction was conducted according to manufacturer'sinstructions.

What is claimed:
 1. A method for generating two or more substantiallymonoclonal populations of template polynucleotides, comprising: (a)contacting single-stranded template polynucleotides with a plurality ofsupports wherein each support comprises a plurality of first primersunder annealing conditions optimized to generate supports having onlyone single-stranded template polynucleotide attached by hybridizationthereto, and optionally extending the first primer in atemplate-dependent polymerization reaction to generate double-strandedtemplate polynucleotides attached to the supports and optionallyseparating the strands of the double-stranded template polynucleotidesattached to the supports; (b) distributing the supports having asingle-stranded template polynucleotide, or optionally a double-strandedtemplate polynucleotide, attached thereto into separate individualreaction sites within an array of reaction sites; and (c) forming two ormore substantially monoclonal nucleic acid populations by amplifying thetemplate polynucleotide at each reaction site, comprising: i) providinga recombinase and a polymerase having a processivity of 100 base pairsor longer, and a second oligonucleotide primer in solution, wherein thesecond oligonucleotide primer in solution comprises an affinity moiety;and ii) clonally amplifying the at least two nucleic acid templates toform at least two substantially monoclonal populations of nucleic acidswherein at least 50% of the nucleic acids in each substantiallymonoclonal population share at least 80% sequence identity wherein thereaction sites are in continuous liquid phase communication with eachother during the amplifying and wherein the continuous liquid phasefurther comprises a binding partner that interacts with the affinitymoiety.
 2. The method of claim 1, wherein the polymerase is a T5 or T7DNA polymerase having reduced exonuclease activity compared to wild-typeT5 or T7 polymerase, and wherein if the polymerase is T7 polymerase, thereaction mixture further comprises thioredoxin.
 3. The method of claim2, wherein the polymerase is a T7 DNA polymerase having a reduced 3′-5′exonuclease activity, and wherein the T7 DNA polymerase has an E7A, aD5A, or both an E7A and a D5A mutation, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO:
 1. 4. The method ofclaim 2, wherein the polymerase is a T7 DNA polymerase selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:
 4. 5. Themethod of claim 2, wherein the continuous liquid phase further comprisesa Bsu polymerase or a Sau polymerase.
 6. The method of claim 1, whereinthe continuous liquid phase further comprises a recombinase accessoryprotein.
 7. The method of claim 6, wherein the recombinase is a UvsXprotein and the recombinase accessory protein is a UvsY protein.
 8. Themethod of claim 1, wherein the continuous liquid phase comprises asingle-stranded binding protein.
 9. The method of claim 8, wherein thesingle-stranded binding protein is gp32.
 10. The method of claim 1,wherein the continuous liquid phase further comprises a sieving agent.11. The method of claim 1, wherein the first oligonucleotide primersattached to the plurality of supports have an identical nucleotidesequence.
 12. The method of claim 1, wherein the templatepolynucleotides attached to the supports comprise an affinity moiety.13. The method of claim 1 wherein a binding partner moiety to which theaffinity moiety binds, is attached to a paramagnetic bead, capable toform purification complexes.
 14. The method of claim 13 furthercomprising attraction of the resulting amplification products with amagnet, thereby removing purification complexes.
 15. The method of claim1, wherein the affinity moiety comprises a biotin moiety and the bindingpartner comprises an avidin-like moiety.
 16. The method of claim 1further comprising: (a) loading at least two different templatepolynucleotides into separate individual reaction chambers in an arrayof reaction chambers; (b) performing two different rounds ofamplification within the reaction chambers comprising: i. a firstamplification reaction, wherein the reaction chambers are contacted withone or more reagents comprising a drag compound comprising a receptormoiety, and ii. a second amplification, comprising contacting thereaction chambers with an amplification primer that hybridizes to atleast a portion of one strand of the template polynucleotides andwherein the amplification primer comprises an affinity moiety thatinteracts with the receptor moiety; thereby amplifying the templatepolynucleotides within the reaction chambers and forming at least twosubstantially monoclonal nucleic acid populations, wherein the reactionchambers are in fluid communication with each other during amplifying.17. The method of claim 16 wherein the affinity moiety comprises biotinand the receptor moiety comprises an avidin-like moiety.
 18. The methodof claim 16 wherein the second amplification does not include contactingreaction chambers with a drag compound.
 19. The method of claim 16wherein a binding partner moiety to which the affinity moiety binds, isattached to a paramagnetic bead, capable to form purification complexes.20. The method of claim 19 further comprising attraction of theresulting amplification products with a magnet, thereby removingpurification complexes.